Antibodies specific for cll-1

ABSTRACT

Provided herein are antibodies specific for CLL-1.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.14/853,881, filed Sep. 14, 2015, which is a continuation of U.S. patentapplication Ser. No. 13/794,525, filed Mar. 11, 2013, which claimspriority to U.S. Provisional Application No. 61/643,739, filed May 7,2012, and U.S. Provisional Application No. 61/699,134, filed Sep. 10,2012, the disclosures of which are incorporated by reference in theirentireties.

REFERENCE TO SUBMISSION OF A SEQUENCE LISTING

This application includes a Sequence Listing as a text file named“092950-0950734 SEQ” created Sep. 9, 2015 and containing 45,885 bytes.The material contained in this text file is incorporated by reference inits entirety for all purposes.

BACKGROUND OF THE INVENTION

C type Lectin Like molecule 1 (CLL-1) is expressed on AML cells, and oncancer stem cells (CSCs), which are cells that can give rise toadditional cancer cells.

One of the major limitations of chemotherapy is the general inability ofanticancer drugs to discriminate between normal and cancer cells. Almostall members of the major categories of antineoplastic agents haveconsiderable toxicity for normal cells.

Compositions that specifically target cancer cells can avoid thisproblem. However, existing cancer targets do not target CSCs. For thisreason, existing chemotherapeutic strategies, even when specificallydelivered to cancer cells, do not effectively eliminate the cancer. Riskof recurrence remains because the surviving CSCs can give rise to newcancer cells.

CSCs express CD34, similar to hematopoietic stem cells (HSCs), but CLL-1is not expressed on HSCs. This allows CSCs to be specifically targetedusing CLL-1. Provided herein are CLL-1 antibodies that recognize a highpercentage of CLL-1 expressing cells. The present CLL-1 antibodies areeffective for both complement dependent and antibody dependentcytoxicity of CLL-1 expressing cells, and inhibit tumor growth of CLL-1expressing cancer cells. The presently described antibodies providenovel diagnostic and therapeutic strategies for targetingCLL-1-associated disorders.

BRIEF SUMMARY OF THE INVENTION

Provided herein are antibodies specific for CLL-1 (“CLL-1 antibodies”)that bind a high percentage of CLL-1 expressing primary cells from AMLpatient samples. In some embodiments, the CLL-1 antibody specificallybinds the extracellular domain of human CLL-1 with a Kd of 10 nM orless, e.g., any of 5 nM, 1 nM, 500 pM, 200 pM, 100 pM, 50 pM or less. Insome embodiments, the CLL-1 antibody binds cynomolgus CLL-1 with a Kd of100 nM, 10 nM, 1 nM, 100 pM or less. For example, in some embodiments,cynomolgus CLL-1 and human CLL-1 compete for binding to the CLL-1antibody. One of skill will understand that higher affinity binding isexpressed as lower Kd (lower concentration of antibody target necessaryfor binding).

In some embodiments, the CLL-1 antibody binds a polypeptide consistingof the C-lectin domain of CLL-1 with a Kd at least 5-fold higher than apolypeptide comprising or consisting of the C-lectin and stalk domainsof CLL-1, e.g., at least any of 10, 20, 50, or 100-fold higher. That is,the antibody binds a polypeptide comprising the stalk and C-lectindomains of CLL-1 with a higher affinity than it binds the C-lectindomain alone or the stalk domain alone. In some embodiments, the CLL-1antibody binds an epitope that includes part of the stalk and part ofthe C-lectin domains. In some embodiments, the CLL-1 antibody binds apolypeptide consisting of the C-lectin and stalk domains of human C-typelectin like molecule (CLL-1) with greater affinity than it binds either(a) a polypeptide consisting of the C-lectin domain of human CLL-1 or(b) a polypeptide consisting of the stalk domain of human CLL-1. Forexample, the CLL-1 antibodies designated as M26 and M31 bind amino acids101-265 of human CLL-1 with higher affinity than amino acids 141-265 ofhuman CLL-1 (with reference to SEQ ID NO:2).

In some embodiments, the CLL-1 antibody binds the C-lectin domain ofCLL-1 with a Kd at least 5-fold higher than it binds the full lengthCLL-1 extracellular domain, e.g., at least any of 10, 20, 50, or100-fold higher. That is, the affinity of the CLL-1 antibody is at leastany of 5, 10, 20, 50, or 100 fold lower than for the full length CLL-1extracellular domain (e.g., as expressed on a cell). In someembodiments, the CLL-1 antibody binds quiescent CLL-1 expressing cells.In some embodiments, the CLL-1 antibody binds quiescent CLL-1 expressingcells with a Kd of 10 nM or less, e.g., any of 1 nM, 500 pM, 200 pM, 100pM, 50 pM or less.

In some embodiments, the CLL-1 antibody binds at least 60% of the cellsin a culture of HL60 cells, e.g., at least any of 70, 75, 80, 85, 90,95, or higher % of the HL60 cells. In some embodiments, the CLL-1antibody binds at least 30% of the nucleated cells in a sample ofprimary cells from an AML patient (e.g., any of 40, 50, 60, 70, 80, 85,90, 95 or higher %), wherein the sample of primary cells is peripheralblood or biopsy of tumor tissue. One of skill will understand that, insuch a cell binding assay, an appropriate concentration of antibody isadded, e.g., so that there are sufficient antibody molecules present tobind the number of cells in the culture or sample.

In some embodiments, the CLL-1 antibody has an EC50 of less than 1 nM inan antibody drug conjugate (ADC) cytotoxicity assay with CLL-1expressing cells, e.g., HL60 cells or primary AML cells. In someembodiments, the EC50 in the ADC assay is any of 500, 200, 100, 50 pM orless. In some embodiments, the CLL-1 antibody reduces colony formationof AML cells by at least 50%, e.g., at least 60%, 70%, 80% or more in anADC cytotoxicity assay.

In some embodiments, the cells are primary patient AML cells. In someembodiments, the cells are AML cancer stem cells. In some embodiments,the CLL-1 antibody does not affect normal CD34+ hematopoietic stem cells(HSCs), or significantly reduce colony formation of normal CD34+ HSCs inan ADC cytotoxicity assay.

In some embodiments, the CLL-1 antibody has an EC50 of 1 ug/ml or lessin a complement dependent cytotoxicity (CDC) assay with CLL-1 expressingcells, e.g., HL60 cells or primary AML cells. In some embodiments, theEC50 in the CDC assay is any of 500, 200, 100, 50, 20, 10 ng/ml or less.In some embodiments, the CLL-1 antibody has an EC50 of 1 ug/ml or lessin an antibody dependent cell-mediated cytotoxicity (ADCC) assay withCLL-1 expressing cells, e.g., CLL-1 transfected 293 cells, HL60 cells,or primary AML cells. In some embodiments, the EC50 in the ADCC assay isany of 500, 200, 100 ng/ml or less. In some embodiments, the CLL-1antibody, when administered to a mouse carrying an AML xenograft for atleast 4 weeks reduces tumor burden at least 10-fold compared to anuntreated control (i.e., a mouse carrying the AML xenograft but nottreated with the CLL-1 antibody). In some embodiments, the AML xenograftis from a human AML cell line, e.g., HL60 or OCI AML-5 cells. In someembodiments, the AML xenograft is from primary human or primate (e.g.,cynomolgus) AML cells.

In some embodiments, the CLL-1 antibody is selected from the groupconsisting of an antibody that competes for binding to CLL-1 (e.g., aCLL-1 expressing cell or AML cell) with an antibody selected from thegroup consisting of:

-   -   an antibody comprising the heavy and light chain CDRs of M26        (see Example 1);    -   an antibody comprising the heavy and light chain CDRs of M31;    -   an antibody comprising the heavy and light chain CDRs of G4;    -   an antibody comprising the heavy and light chain CDRs of M22;    -   an antibody comprising the heavy and light chain CDRs of M29;    -   an antibody comprising the heavy and light chain CDRs of M2;    -   an antibody comprising the heavy and light chain CDRs of M5;    -   an antibody comprising the heavy and light chain CDRs of G12;    -   an antibody comprising the heavy and light chain CDRs of M41;    -   an antibody comprising the heavy and light chain CDRs of E3;    -   an antibody comprising the heavy and light chain CDRs of B10;    -   an antibody comprising the heavy and light chain CDRs of G2;    -   an antibody comprising the heavy and light chain CDRs of G6;    -   an antibody comprising the heavy and light chain CDRs of G8;    -   an antibody comprising the heavy and light chain CDRs of G10;    -   an antibody comprising the heavy and light chain CDRs of G14;    -   an antibody comprising the heavy and light chain CDRs of G16;    -   an antibody comprising the heavy and light chain CDRs of G23;    -   an antibody comprising the heavy and light chain CDRs of G26;    -   an antibody comprising the heavy and light chain CDRs of G28;        and    -   an antibody comprising the heavy and light chain CDRs of G30.

In some embodiments, the CLL-1 antibody is selected from an antibodyselected from the group consisting of:

-   -   an antibody comprising the heavy and light chain CDRs of M26        (see Example 1);    -   an antibody comprising the heavy and light chain CDRs of M31;    -   an antibody comprising the heavy and light chain CDRs of G4;    -   an antibody comprising the heavy and light chain CDRs of M22;    -   an antibody comprising the heavy and light chain CDRs of M29;    -   an antibody comprising the heavy and light chain CDRs of M2;    -   an antibody comprising the heavy and light chain CDRs of M5;    -   an antibody comprising the heavy and light chain CDRs of G12;    -   an antibody comprising the heavy and light chain CDRs of M41;    -   an antibody comprising the heavy and light chain CDRs of E3;    -   an antibody comprising the heavy and light chain CDRs of B10;    -   an antibody comprising the heavy and light chain CDRs of G2;    -   an antibody comprising the heavy and light chain CDRs of G6;    -   an antibody comprising the heavy and light chain CDRs of G8;    -   an antibody comprising the heavy and light chain CDRs of G10;    -   an antibody comprising the heavy and light chain CDRs of G14;    -   an antibody comprising the heavy and light chain CDRs of G16;    -   an antibody comprising the heavy and light chain CDRs of G23;    -   an antibody comprising the heavy and light chain CDRs of G26;    -   an antibody comprising the heavy and light chain CDRs of G28;        and    -   an antibody comprising the heavy and light chain CDRs of G30,        wherein any one or more of the the selected CDRs can have 1, 2,        or 3 conservative amino acid substitutions compared to the        original CDR sequence.

In some embodiments, the CLL-1 antibody comprises the heavy and lightchain CDRs of M26. In some embodiments, the CLL-1 antibody comprises theheavy and light chain CDRs of M31. In some embodiments, the CLL-1antibody comprises the heavy and light chain CDRs of G4.

In some embodiments, the CLL-1 antibody as described above binds apolypeptide consisting of the C-lectin domain of CLL-1 with a Kd atleast 5-fold higher than a polypeptide consisting of the C-lectin andstalk domains of CLL-1 (e.g., any of 10, 20, 50, 100 or higher fold). Insome embodiments, the CLL-1 antibody binds the C-lectin domain of CLL-1with a Kd at least 5-fold higher than it binds full length CLL-1extracellular domain (e.g., any of 10, 20, 50, 100 or higher fold). Insome embodiments, the CLL-1 antibody as described above further binds atleast 80% of the cells in a culture of HL60 cells (e.g., any of 85, 90,95 or higher %). In some embodiments, the CLL-1 antibody as describedabove further binds at least 30% of the nucleated cells in a sample ofAML cells from an individual with AML (e.g., any of 40, 50, 60, 70, 80,85, 90, 95, or higher %). Again, in such a cell binding assay, anappropriate concentration of antibody is added, e.g., so that there aresufficient antibody molecules present to bind the number of cells in theculture or sample, and antibody concentration is not the limitingfactor.

In some embodiments, the CLL-1 antibody as described above is a chimericantibody with a human Fc region, e.g., from IgG1. In some embodiments,the CLL-1 antibody as described above is humanized. In some embodiments,the CLL-1 antibody as described above is an Fv fragment (e.g., Fab,Fab′, or F(ab′)2). In some embodiments, the CLL-1 antibody as describedabove is labeled e.g., conjugated to a detectable moiety. In someembodiments, the CLL-1 as described above is attached to a therapeuticcompound, e.g., a cytotoxin or cell growth inhibitor.

In some embodiments, the CLL-1 antibody is selected from the groupconsisting of:

-   -   an antibody comprising variable region sequences with        substantial identity (at least any of 85, 90, 95, or 98%        identity) to those of M26 (Vh=SEQ ID NO:4; V1=SEQ ID NO:6)    -   an antibody comprising variable region sequences with        substantial identity to those of M31 (Vh=SEQ ID NO:8; V1=SEQ ID        NO:10);    -   an antibody comprising variable region sequences with        substantial identity to those of G4 (Vh=SEQ ID NO:12; V1=SEQ ID        NO:14);    -   an antibody comprising variable region sequences with        substantial identity to those of M22 (Vh=SEQ ID NO:16; V1=SEQ ID        NO:18);    -   an antibody comprising variable region sequences with        substantial identity to those of M29 (Vh=SEQ ID NO:20; V1=SEQ ID        NO:22);    -   an antibody comprising variable region sequences with        substantial identity to those of M2 (Vh=SEQ ID NO:24; V1=SEQ ID        NO:26);    -   an antibody comprising variable region sequences with        substantial identity to those of M5 (Vh=SEQ ID NO:28; V1=SEQ ID        NO:30);    -   an antibody comprising variable region sequences with        substantial identity to those of G12 (Vh=SEQ ID NO:32; V1=SEQ ID        NO:34)    -   an antibody comprising variable region sequences with        substantial identity to those of M41;    -   an antibody comprising variable region sequences with        substantial identity to those of E3;    -   an antibody comprising variable region sequences with        substantial identity to those of B10;    -   an antibody comprising variable region sequences with        substantial identity to those of G2;    -   an antibody comprising variable region sequences with        substantial identity to those of G6;    -   an antibody comprising variable region sequences with        substantial identity to those of G8;    -   an antibody comprising variable region sequences with        substantial identity to those of G10;    -   an antibody comprising variable region sequences with        substantial identity to those of G14;    -   an antibody comprising variable region sequences with        substantial identity to those of G16;    -   an antibody comprising variable region sequences with        substantial identity to those of G23;    -   an antibody comprising variable region sequences with        substantial identity to those of G26;    -   an antibody comprising variable region sequences with        substantial identity to those of G28; and    -   an antibody comprising variable region sequences with        substantial identity to those of G30.

In some embodiments, the substantially identical antibody has the CDRsequences of the original antibody.

In some embodiments, the CLL-1 antibody competes for binding with anantibody selected from the group consisting of:

-   -   an antibody comprising variable region sequences of M26 (Vh=SEQ        ID NO:4; V1=SEQ ID NO:6)    -   an antibody comprising variable region sequences of M31 (Vh=SEQ        ID NO:8; V1=SEQ ID NO:10);    -   an antibody comprising variable region sequences of G4 (Vh=SEQ        ID NO:12; V1=SEQ ID NO:14);    -   an antibody comprising variable region sequences of M22 (Vh=SEQ        ID NO:16; V1=SEQ ID NO:18);    -   an antibody comprising variable region sequences of M29 (Vh=SEQ        ID NO:20; V1=SEQ ID NO:22);    -   an antibody comprising variable region sequences of M2 (Vh=SEQ        ID NO:24; V1=SEQ ID NO:26);    -   an antibody comprising variable region sequences of M5 (Vh=SEQ        ID NO:28; V1=SEQ ID NO:30);    -   an antibody comprising variable region sequences of G12 (Vh=SEQ        ID NO:32; V1=SEQ ID NO:34)    -   an antibody comprising variable region sequences of M41;    -   an antibody comprising variable region sequences of E3;    -   an antibody comprising variable region sequences of B10;    -   an antibody comprising variable region sequences of G2;    -   an antibody comprising variable region sequences of G6;    -   an antibody comprising variable region sequences of G8;    -   an antibody comprising variable region sequences of G10;    -   an antibody comprising variable region sequences of G14;    -   an antibody comprising variable region sequences of G16;    -   an antibody comprising variable region sequences of G23;    -   an antibody comprising variable region sequences of G26;    -   an antibody comprising variable region sequences of G28; and    -   an antibody comprising variable region sequences of G30.

In some embodiments, the CLL-1 antibody is selected from the groupconsisting of:

-   -   an antibody comprising variable region sequences of M26 (Vh=SEQ        ID NO:4; V1=SEQ ID NO:6)    -   an antibody comprising variable region sequences of M31 (Vh=SEQ        ID NO:8; V1=SEQ ID NO:10);    -   an antibody comprising variable region sequences of G4 (Vh=SEQ        ID NO:12; V1=SEQ ID NO:14);    -   an antibody comprising variable region sequences of M22 (Vh=SEQ        ID NO:16; V1=SEQ ID NO:18);    -   an antibody comprising variable region sequences of M29 (Vh=SEQ        ID NO:20; V1=SEQ ID NO:22);    -   an antibody comprising variable region sequences of M2 (Vh=SEQ        ID NO:24; V1=SEQ ID NO:26);    -   an antibody comprising variable region sequences of M5 (Vh=SEQ        ID NO:28; V1=SEQ ID NO:30);    -   an antibody comprising variable region sequences of G12 (Vh=SEQ        ID NO:32; V1=SEQ ID NO:34)    -   an antibody comprising variable region sequences of M41;    -   an antibody comprising variable region sequences of E3;    -   an antibody comprising variable region sequences of B10;    -   an antibody comprising variable region sequences of G2;    -   an antibody comprising variable region sequences of G6;    -   an antibody comprising variable region sequences of G8;    -   an antibody comprising variable region sequences of G10;    -   an antibody comprising variable region sequences of G14;    -   an antibody comprising variable region sequences of G16;    -   an antibody comprising variable region sequences of G23;    -   an antibody comprising variable region sequences of G26;    -   an antibody comprising variable region sequences of G28; and    -   an antibody comprising variable region sequences of G30.

In some embodiments, the CLL-1 antibody comprises the heavy and lightchain variable region sequences of M26. In some embodiments, the CLL-1antibody comprises the heavy and light chain variable region sequencesof M31. In some embodiments, the CLL-1 antibody comprises the heavy andlight chain variable region sequences of G4.

In some embodiments, the CLL-1 antibody as described binds a polypeptideconsisting of the C-lectin domain of CLL-1 with a Kd at least 5-foldhigher than a polypeptide consisting of the C-lectin and stalk domainsof CLL-1 (e.g., any of 10, 20, 50, 100 or higher fold). In someembodiments, the CLL-1 antibody binds the C-lectin domain of CLL-1 witha Kd at least 5-fold higher than it binds full length CLL-1extracellular domain (e.g., any of 10, 20, 50, 100 or higher fold). Insome embodiments, the CLL-1 antibody as described above further binds atleast 80% of the cells in a culture of HL60 cells (e.g., any of 85, 90,95 or higher %). In some embodiments, the CLL-1 antibody as describedabove further binds at least 30% of the nucleated cells in a sample ofAML cells from an individual with AML (e.g., any of 40, 50, 60, 70, 80,85, 90, 95, or higher %).

In some embodiments, the CLL-1 antibody as described above is an Fvfragment (e.g., Fab, Fab′, or F(ab′)2). In some embodiments, theantibody comprises two distinct variable regions, with two distinctepitope binding sequences, in a single antibody construct (e.g., withone epitope binding region from M26, M31, G4, or M22 and one epitopebinding region from M26, M31, G4, or M22 in any combination). In someembodiments, the CLL-1 antibody as described above is labeled, e.g.,conjugated to a detectable moiety. In some embodiments, the CLL-1 asdescribed above is attached to a therapeutic compound, e.g., a cytotoxinor cell growth inhibitor.

Further provided are pharmaceutical compositions comprising a CLL-1antibody as described herein and a pharmaceutically acceptable excipientor carrier.

Provided are methods for determining whether a cell expresses CLL-1comprising: contacting a CLL-1 antibody (i.e., a CLL-1 antibody havingany of the activities or sequences described above) with the cell;detecting binding of the antibody to the cell, wherein binding of theantibody to the cell indicates that the cell expresses CLL-1; anddetermining whether the cell expresses CLL-1. In some embodiments, themethod further comprises determining whether the cell expresses CD34. Insome embodiments, the method further comprises determining whether thecell expresses CD38. In some embodiments, the method further comprisesdetermining whether the cell expresses CD45. In some embodiments, thecell is in a biological sample obtained from an individual (e.g., ablood sample or a biopsy from a tumor or tissue). In some embodiments,antibody binding is detected by FACS.

Also provided are methods of identifying a myeloid cancer cell (a CLL-1expresssing cancer cell, e.g., from a myeloproliferative disorder suchas AML, CML, CMML, multiple myeloma, plasmacytoma, or MDS) or a CSC(e.g., LSC or myeloid cancer cell blast) comprising: contacting a CLL-1antibody (i.e., a CLL-1 antibody having any of the activities orsequences described above) with a cell; detecting binding of theantibody to the cell; and identifying a CSC or myeloid cancer cell whenthe antibody binds the cell. In some embodiments, the myeloid cancercell is selected from an AML, CIVIL, CMML, multiple myeloma,plasmacytoma, or MDS cell. In some embodiments, the method furthercomprises determining whether the cell expresses CD45 and identifying anAML cell when the cell expresses CD45. In some embodiments, the methodfurther comprises determining whether the cell expresses CD34 andidentifying a CSC when the cell expresses CD34. In some embodiments, thecell is in a biological sample from an individual. In some embodiments,antibody binding is detected by FACS.

Further provided are methods of diagnosing an individual for amyeloproliferative disorder (e.g., AML, CIVIL, MDS, CMML, multiplemyeloma, plasmacytoma myelofibrosis) comprising contacting a CLL-1antibody (i.e., a CLL-1 antibody having any of the activities orsequences described above) with a biological sample from the individual;detecting binding of the antibody to a cell in the biological sample;and diagnosing the individual with a myeloproliferative disorder whenthe antibody binds the cell. In some embodiments, the biological sampleis a blood sample (e.g., peripheral nucleated blood cells) or biopsyfrom a tumor or tissue. In some embodiments, the method furthercomprises determining whether the cell expresses CD34. In someembodiments, the method further comprises determining a course oftreatment for the individual when a myeloproliferative disorder isdiagnosed. In some embodiments, the course of treatment includesadministration of an effective dose of a CLL-1 antibody. In someembodiments, the effective dose of the CLL-1 antibody is administered ina pharmaceutical composition comprising a pharmaceutically acceptableexcipient. In some embodiments, the method further comprises monitoringthe individual, e.g., when a myeloproliferative disorder is diagnosed,or when the individual has been previously diagnosed with amyeloproliferative disorder but received treatment for the disease.

Additionally provided are methods of inhibiting survival of a CLL-1expressing cell (e.g., reducing cell growth or division, mediating ADC,mediating CDC) comprising contacting a CLL-1 antibody (i.e., a CLL-1antibody having any of the activities or sequences described above) withthe cell and inhibiting survival of the cell. In some embodiments, thecontacting comprises administering the antibody (e.g., in apharmaceutical composition) to an individual, e.g., an individualdiagnosed with a myeloproliferative disorder (e.g., AML, CIVIL, MDS,CMML, multiple myeloma, plasmacytoma myelofibrosis). In someembodiments, the CLL-1 antibody is administered in a dose effective toinhibit survival of CLL-1 expressing cells.

Provided are methods of treating a myeloproliferative disorder in anindividual (e.g., reducing tumor growth or engraftment compared to anuntreated control) comprising administering an effective dose of CLL-1antibody (i.e., a CLL-1 antibody having any of the activities orsequences described above) to the individual, thereby treating themyeloproliferative disorder in the individual. In some embodiments, themyeloproliferative disorder is selected from AML, CML, MDS, CMML,multiple myeloma, plasmacytoma, and myelofibrosis. In some embodiments,the effective dose of the CLL-1 antibody is administered in apharmaceutical composition comprising a pharmaceutically acceptableexcipient. In some embodiments, the individual has been diagnosed with amyeloproliferative disorder, e.g., using a CLL-1 antibody as describedherein. In some embodiments, the method of treatment further comprisesmonitoring cell growth (e.g., tumor growth or circulating myeloid cancercells) in the individual, e.g., using a CLL-1 antibody as describedherein. In some embodiments, the CLL-1 antibody is attached to atherapeutic compound, e.g., a cytotoxin or cell growth inhibitor.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C show the results of complement dependent cytotoxicity (CDC)assays using primary cells from 3 different AML patients, #49 (FIG. 1A),#50 (FIG. 1B), and #52 (FIG. 1C). A and B show that CLL-1 antibody cloneM26 has an EC50 between 10 and 100 ng/mL. FIG. 1C shows the results forCLL-1 antibody clones M26, M31, and negative controls E12 (unrelatedantibody) and IgG. M26 and M31 both have an EC50 between 10 and 100ng/mL.

FIG. 2 shows the antitumor effect of CLL-1 antibody clones in a mousexenograft model. HL60 AML cells were injected subcutaneously into mice.Mice were divided into 5 groups, with n=6 mice per group: (1) IgG2acontrol; (2) M5; (3) M13; (4) M26; and (5) M31. Mice received 200 ugantibody once per week for 7 weeks. P<0.05 vs control for all treatmentgroups.

FIG. 3 shows the antitumor effect of CLL-1 antibody clones in a mouseorthotopic xenograft model. AML cells were injected intravenously intoimmunocompromised NSG (NOD/SCID/IL2 receptor Gamma chain knockout) mice.Mice were divided into 5 groups, with n=6 mice per group: (1) IgG2acontrol; (2) M5; (3) M13; (4) M26; and (5) M31. Mice received 200 ugantibody twice per week for 2 weeks, and were sacrificed 4 weekspost-transplant. Tumor burden (CD45+ CLL-1+ cells) in bone marrow wasdetermined by FACS.

FIGS. 4A-4B show that CLL-1 Antibody Drug Conjugates (ADC) inhibitcolony formation by AML stem cells but not normal hematopoietic stemcells (CD34+ HSCs). FIG. 4A shows that seeded CD34+ HSCs form coloniesin the presence of antibody drug conjugates at a level of the negativecontrol (no antibody or antibody drug conjugate). FIG. 4B shows that,for seeded total PBMC, AML cancer stem cells (CSCs) have 80% less colonyformation in the presence of CLL-1 antibody M26 conjugated to saporincompared to the negative control (no antibody or antibody drugconjugate).

FIG. 5 shows that CLL-1 antibody clones M26, M31, and G4 (also labeled31.G4) bind to human PBMCs in both mouse and chimeric human (Chi) forms.Negative controls include the IgG corresponding to each CLL-1 antibody,but specific for an unrelated antigen. Mononuclear cells were separatedfrom PBMC samples, and FACS was used to characterize the cells accordingto expression of CD89 (granulocytes), CD14 (monocytes and granulocytes),CD3 (lymphocytes), and CD19 (B cells). The percentage of CLL-1 positivestaining for each population is shown in that order from left to rightfor each CLL-1 antibody.

FIG. 6 shows that CLL-1 antibody clones M26, M31, and G4 (labeled 31G4)bind to cynomolgus PBMCs in both orginal mouse and chimeric human (Chi)forms. Negative controls include the IgG corresponding to each CLL-1antibody, but specific for an unrelated antigen. Mononuclear cells wereseparated from PBMC samples, and FACS was used to characterize the cellsaccording to expression of CD3 (lymphocytes), CD19 (B cells), CD14(granulocytes), CD14 (monocytes), and CD89 (granulocytes). Thepercentage of CLL-1 positive staining for each population is shown inthat order from left to right for each CLL-1 antibody.

FIGS. 7A-7B show that both mouse and chimeric human CLL-1 have AntibodyDrug Conjugate (ADC) activity on CLL-1 transfected 293 cells in vitro.FIG. 7A shows results for mouse CLL-1 antibody clones M26, M31, and G4(31G4) compared to a negative control mouse IgG2a. FIG. 7B shows resultsfor the corresponding chimeric human CLL-1 antibody clones.

FIG. 8 shows that chimeric human CLL-1 antibody clones M26, M31, and G4(31G4) mediate antibody-dependent cell-mediated cytotoxicity (ADCC) onCLL-1 transfected 293 cells. The EC₅₀ (ng/ml) for ChiM26, ChiM31, andChi31G4 is 79, 143, and 105, respectively.

FIGS. 9A-9B show the antitumor effect of CLL-1 antibody clones in amouse xenograft model. NOD/SCID mice were irradiated on Day −1, and onDay 0, HL60 cells were injected into the tail veins (3×10⁶ cells permouse). Mice were divided into 3 groups, with n=6 mice per group: (1)huIgG control; (2) M26; (3) ChiM31. Mice received 8 antibody injections(200 ug) over the course of 22 days, and were sacrificed on day 26.Tumor burden in bone marrow was determined by FACS. FIG. 9A shows thepercent huCD45+CD33+ AML cells, and FIG. 9B shows the percent huCD45+CLL-1+ AML CSCs.

FIGS. 10A-10B show the antitumor effect of CLL-1 antibody clones in amouse xenograft model. NOD/SCID mice were irradiated on Day −1, and onDay 0, OCI AML-5 cells were injected into the tail veins (5×10⁶ cellsper mouse). Mice were divided into 5 groups, with n=6 mice per group:(1) huIgG control; (2) M26; (3) ChiM26; (4) ChiM31; (5) ChiG4. Micereceived 8 antibody injections (200 ug) over the course of 19 days, andwere sacrificed on day 24 post. Tumor burden in bone marrow wasdetermined by FACS. FIG. 10A shows the percentage of huCD45+CD33+ AMLcells. FIG. 10B shows the log₁₀ percentage of huCD45+CD33+ AML cells, toobserve better resolution between the results. The data show that all 4CLL-1 antibodies tested effectively reduced tumor burden, and that M26,ChiM26, and ChiM31 had the greatest antitumor effect.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

Provided herein are antibodies specific for CLL-1 with variousadvantageous properties. Such antibodies were selected based on at leastone of the following criteria:

-   -   Affinity for human CLL-1 in the picomolar to nanomolar range;    -   Binding to a relatively high percentage of samples obtained from        AML patients (e.g., a higher percentage of AML patients than the        X357 or X1057 CLL-1 antibody, or at least 50% of AML patient        samples);    -   Binding to a relatively high percentage of cells (e.g.,        peripheral blood mononuclear cells (PBMCs)) in an AML patient        sample (e.g., a higher percentage of cells than the X357 or        X1057 CLL-1 antibody, or at least 50% of the cells in an AML        patient sample);    -   Active in antibody drug conjugate (ADC) cytotoxicity assay;    -   Active in complement dependent cytotoxicity (CDC) assay;    -   Active in antibody dependent cell cytotoxicity (ADCC) assay;    -   Antitumor activity, in vitro or in vivo (xenograft mouse model);    -   Specific binding to, and ADC activity in AML cells, but not        normal HSCs;    -   Binding to species homolog of an animal model (e.g., cynomolgus        CLL-1);    -   Above activities retained for antibodies in chimeric human form.

The presently described CLL-1 antibodies do not all have all of theselective characteristics, but are further described, e.g., according tosequence, below. The present CLL-1 antibodies can be used for detectionof CLL-1 expressing cells, e.g., for diagnosis or monitoring of CLL-1expressing cancer cells in an individual, or for treatment of CLL-1expressing cancer such as AML.

II. Definitions

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by a person of ordinaryskill in the art. See, e.g., Lackie, DICTIONARY OF CELL AND MOLECULARBIOLOGY, Elsevier (4^(th) ed. 2007); Sambrook et al., MOLECULAR CLONING,A LABORATORY MANUAL, Cold Springs Harbor Press (Cold Springs Harbor, N Y1989). The term “a” or “an” is intended to mean “one or more.” The term“comprise” and variations thereof such as “comprises” and “comprising,”when preceding the recitation of a step or an element, are intended tomean that the addition of further steps or elements is optional and notexcluded. Any methods, devices and materials similar or equivalent tothose described herein can be used in the practice of this invention.The following definitions are provided to facilitate understanding ofcertain terms used frequently herein and are not meant to limit thescope of the present disclosure.

C-type Lectin-Like molecule 1 (CLL-1), also known as CLEC12A, DCAL-2,and MICL, is a type II membrane protein (ITIM domain—TM domain-stalkdomain-lectin-like domain). The extracellular domain of CLL-1 is highlyglycosylated, and it is expressed exclusively in cells of myeloidlineage. CLL-1 is also expressed on AML, MDS, and CIVIL cells. CLL-1expression can be used to distinguish between normal hematopoietic stemcells (HSCs), which do not express CLL-1, and leukemic stem cells(LSCs), where it is expressed. LSCs are CD34+ cells in leukemia patientsthat lead to production of cancer cells and recurrence of cancer. SeeBakker et al. (2004) Cancer Res. 64:8443.

The nucleotide and protein sequences of CLL-1 are known for manyspecies. For example, the human sequences can be found at Genbankaccession number AF247788.1 (coding sequence shown in SEQ ID NO:1) andUniprot accession number Q5QGZ9 (SEQ ID NO:2). For the human CLL-1protein shown as SEQ ID NO:2, the extracellular domain comprisesapproximately amino acids 65-265, the transmembrane domain comprisesapproximately amino acids 44-64, and the cytoplasmic domain comprisesapproximately amino acids 1-43. The stalk domain of human CLL-1 spansamino acids 65-139, and the C lectin domain spans amino acids 140-249,both with reference to the sequence shown in SEQ ID NO:2. One of skillwill understand that CLL-1 variants (e.g., species homologs, allelicvariants, etc.) can be optimally aligned, e.g., for identification ofconserved residues and domains.

The terms “CLL-1 specific antibody,” “anti-CLL-1 antibody,” “CLL-1antibody,” and “anti-CLL-1” are used synonymously herein to refer to anantibody that specifically binds to CLL-1, including variouslyglycosylated forms of CLL-1. The CLL-1 antibodies described hereinspecifically bind the CLL-1 polypeptide expressed, e.g., on the surfaceof certain cancer cells, but not to HSCs. As discussed in more detailbelow, the present anti-CLL-1 antibodies can bind CLL-1 expressingcells, bind a larger percentage of AML cells compared to otherAML-targeting antibodies, inhibit AML cell proliferation, and mediatetheir destruction.

A “CLL-1 associated disorder” (or CLL-1 related disorder, CLL-1disorder, CLL-1 related condition or disease, etc.) refers to conditionsand diseases correlated with elevated or reduced cell surface expressionof CLL-1 as compared to CLL-1 expression in a standard control (e.g., anormal, non-disease, non-cancer cell). Elevated CLL-1 levels areassociated with cancer cells, in particular, leukemias such as AML(acute myelogenous leukemia), MDS (myelodysplastic syndrome), and CIVIL(chronic myelogenous leukemia), and in hematopoietic CSCs (e.g., LSCs).

The term “antibody” refers to a polypeptide structure, e.g., animmunoglobulin, conjugate, or fragment thereof that retains antigenbinding activity. The term includes but is not limited to polyclonal ormonoclonal antibodies of the isotype classes IgA, IgD, IgE, IgG, andIgM, derived from human or other mammalian cells, including natural orgenetically modified forms such as humanized, human, single-chain,chimeric, synthetic, recombinant, hybrid, mutated, grafted, and in vitrogenerated antibodies. The term encompases conjugates, including but notlimited to fusion proteins containing an immunoglobulin moiety (e.g.,chimeric or bispecific antibodies or scFv's), and fragments, such asFab, F(ab′)2, Fv, scFv, Fd, dAb and other compositions.

An exemplary immunoglobulin (antibody) structural unit comprises atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(V_(L)) and variable heavy chain (V_(H)) refer to these light and heavychains respectively. The variable region contains the antigen-bindingregion of the antibody (or its functional equivalent) and is mostcritical in specificity and affinity of binding. See Paul, FundamentalImmunology (2003).

Antibodies can exist as intact immunoglobulins or as any of a number ofwell-characterized fragments that include specific antigen-bindingactivity. For the sake of clarity, a tetrameric antibody with heavy andlight chains is referred to herein as an “intact immunoglobulin,” andcan be naturally occurring, polyclonal, monoclonal, or recombinantlyproduced. Fragments can be produced by digestion with variouspeptidases. Pepsin digests an antibody below the disulfide linkages inthe hinge region to produce F(ab)′2, a dimer of Fab which itself is alight chain joined to V_(H)-C_(H)1 by a disulfide bond. The F(ab)′2 maybe reduced under mild conditions to break the disulfide linkage in thehinge region, thereby converting the F(ab)′2 dimer into an Fab′ monomer.The Fab′ monomer is essentially Fab with part of the hinge region. Whilevarious antibody fragments are defined in terms of the digestion of anintact antibody, one of skill will appreciate that such fragments may besynthesized de novo either chemically or by using recombinant DNAmethodology. Thus, the term antibody, as used herein, also includesantibody fragments either produced by the modification of wholeantibodies, or those synthesized de novo using recombinant DNAmethodologies or those identified using phage display libraries (see,e.g., McCafferty et al., Nature 348:552-554 (1990)).

As used herein, the term “Fv” refers to a monovalent or bi-valentvariable region fragment, and can encompass only the variable regions(e.g., V_(L) and/or V_(H)), as well as longer fragments, e.g., an Fab,Fab′ or F(ab′)2, which also includes C_(L) and/or C_(H)1. Unlessotherwise specified, the term “Fc” refers to a heavy chain monomer ordimer comprising C_(H)1 and C_(H)2 regions.

A single chain Fv (scFv) refers to a polypeptide comprising a V_(L) andV_(H) joined by a linker, e.g., a peptide linker. ScFvs can also be usedto form tandem (or di-valent) scFvs or diabodies. Production andproperties of tandem scFvs and diabodies are described, e.g., in Asanoet al. (2011) J Biol. Chem. 286:1812; Kenanova et al. (2010) Prot EngDesign Sel 23:789; Asano et al. (2008) Prot Eng Design Sel 21:597.

A “monoclonal antibody” refers to a clonal preparation of antibodieswith a single binding specificity and affinity for a given epitope on anantigen. A “polyclonal antibody” refers to a preparation of antibodiesthat are raised against a single antigen, but with different bindingspecificities and affinities.

As used herein, “V-region” refers to an antibody variable region domaincomprising the segments of Framework 1, CDR1, Framework 2, CDR2, andFramework 3, including CDR3 and Framework 4, which segments are added tothe V-segment as a consequence of rearrangement of the heavy chain andlight chain V-region genes during B-cell differentiation.

As used herein, “complementarity-determining region (CDR)” refers to thethree hypervariable regions in each chain that interrupt the four“framework” regions established by the light and heavy chain variableregions. The CDRs are primarily responsible for binding to an epitope ofan antigen. The CDRs of each chain are typically referred to as CDR1,CDR2, and CDR3, numbered sequentially starting from the N-terminus, andare also typically identified by the chain in which the particular CDRis located. Thus, a V_(H) CDR3 is located in the variable domain of theheavy chain of the antibody in which it is found, whereas a V_(L) CDR1is the CDR1 from the variable domain of the light chain of the antibodyin which it is found.

The sequences of the framework regions of different light or heavychains are relatively conserved within a species. The framework regionof an antibody, that is the combined framework regions of theconstituent light and heavy chains, serves to position and align theCDRs in three dimensional space.

The amino acid sequences of the CDRs and framework regions can bedetermined using various well known definitions in the art, e.g., Kabat,Chothia, international ImMunoGeneTics database (IMGT), and AbM (see,e.g., Johnson et al., supra; Chothia & Lesk, (1987)J Mol. Biol. 196,901-917; Chothia et al. (1989) Nature 342, 877-883; Chothia et al.(1992) J. Mol. Biol. 227, 799-817; Al-Lazikani et al., J. Mol. Biol1997, 273(4)). A helpful guide for locating CDRs using the Kabat systemcan be found at the website available at bioinf.org.uk/abs. Definitionsof antigen combining sites are also described in the following: Ruiz etal. Nucleic Acids Res., 28, 219-221 (2000); and Lefranc Nucleic AcidsRes. January 1; 29(1):207-9 (2001); MacCallum et al., J. Mol. Biol.,262: 732-745 (1996); and Martin et al, Proc. Natl Acad. Sci. USA, 86,9268-9272 (1989); Martin, et al, Methods Enzymol., 203: 121-153, (1991);Pedersen et al, Immunomethods, 1, 126, (1992); and Rees et al, InSternberg M. J. E. (ed.), Protein Structure Prediction. OxfordUniversity Press, Oxford, 141-172 1996).

A “chimeric antibody” refers to an antibody in which (a) the constantregion, or a portion thereof, is altered, replaced or exchanged so thatthe antigen binding site (variable region, CDR, or portion thereof) islinked to a constant region of a different or altered class, effectorfunction and/or species; or (b) the variable region, or a portionthereof, is altered, replaced or exchanged with a variable region havinga different or altered antigen specificity (e.g., CDR and frameworkregions from different species). Chimeric antibodies can includevariable region fragments, e.g., a recombinant antibody comprising twoFab or Fv regions or an scFv. A chimeric can also, as indicated above,include an Fc region from a different source than the attached Fvregions. In some cases, the chimeric antibody includes chimerism withinthe Fv region. An example of such a chimeric antibody would be ahumanized antibody where the FRs and CDRs are from different sources.

Humanized antibodies are antibodies in which the antigen binding loops,i.e., CDRs, obtained from the V_(H) and V_(L) regions of a non-humanantibody are grafted to a human framework sequence. Humanization, i.e.,substitution of non-human CDR sequences for the corresponding sequencesof a human antibody, can be performed following the methods describedin, e.g., U.S. Pat. Nos. 5,545,806; 5,569,825; 5,633,425; 5,661,016;Riechmann et al., Nature 332:323-327 (1988); Marks et al.,Bio/Technology 10:779-783 (1992); Morrison, Nature 368:812-13 (1994);Fishwild et al., Nature Biotechnology 14:845-51 (1996). Transgenic mice,or other organisms such as other mammals, may also be used to expresshumanized or human antibodies, as disclosed in U.S. Pat. No. 6,673,986.

The terms “antigen,” “immunogen,” “antibody target,” “target analyte,”and like terms are used herein to refer to a molecule, compound, orcomplex that is recognized by an antibody, i.e., can be specificallybound by the antibody. The term can refer to any molecule that can bespecifically recognized by an antibody, e.g., a polypeptide,polynucleotide, carbohydrate, lipid, chemical moiety, or combinationsthereof (e.g., phosphorylated or glycosylated polypeptides, etc.). Oneof skill will understand that the term does not indicate that themolecule is immunogenic in every context, but simply indicates that itcan be targeted by an antibody.

Antibodies bind to an “epitope” on an antigen. The epitope is thelocalized site on the antigen that is recognized and bound by theantibody. Epitopes can include a few amino acids or portions of a fewamino acids, e.g., 5 or 6, or more, e.g., 20 or more amino acids, orportions of those amino acids. In some cases, the epitope includesnon-protein components, e.g., from a carbohydrate, nucleic acid, orlipid. In some cases, the epitope is a three-dimensional moiety. Thus,for example, where the target is a protein, the epitope can be comprisedof consecutive amino acids, or amino acids from different parts of theprotein that are brought into proximity by protein folding (e.g., adiscontinuous epitope). The same is true for other types of targetmolecules that form three-dimensional structures.

The terms “specific for,” “specifically binds,” and like terms refer toa molecule (e.g., antibody or antibody fragment) that binds to a targetwith at least 2-fold greater affinity than non-target compounds, e.g.,at least any of 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold,20-fold, 25-fold, 50-fold, or 100-fold greater affinity. For example, anantibody that specifically binds a primary antibody will typically bindthe primary antibody with at least a 2-fold greater affinity than anon-primary antibody target (e.g., an antibody from a different speciesor of a different isotype, or a non-antibody target).

The term “binds” with respect to an antibody target (e.g., antigen,analyte, immune complex), typically indicates that an antibody binds amajority of the antibody targets in a pure population (assumingappropriate molar ratios). For example, an antibody that binds a givenantibody target typically binds to at least 2/3 of the antibody targetsin a solution (e.g., at least any of 75, 80, 85, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, or 100%). One of skill will recognize that somevariability will arise depending on the method and/or threshold ofdetermining binding.

The term “cross-linked” with respect to an antibody refers to attachmentof the antibody to a solid or semisolid matrix (e.g., sepharose, beads,culture plate), or to another protein or antibody. For example, theantibody can be multimerized to create an antibody complex with multiple(more than 2) antigen-binding sites. The antibody can be multimerized byexpressing the antibody as a high-valency isotype (e.g., IgA or IgM,which typically form complexes of 2 or 5 antibodies, respectively).Antibody multimerization can also be carried out by using a cross-linkercomprising a reactive group capable of linking proteins (e.g.,carbodiimide, NHS esters, etc). Methods and compositions forcross-linking an antibody to a matrix are described, e.g., in the Abcamand New England Biolab catalogs and websites (available at abcam.com andneb.com). Cross-linker compounds with various reactive groups aredescribed, e.g., in Thermo Fisher Scientific catalog and website(available at piercenet.com).

As used herein, a first antibody, or an antigen-binding portion thereof,“competes” for binding to a target with a second antibody, or anantigen-binding portion thereof, when binding of the second antibodywith the target is detectably decreased in the presence of the firstantibody compared to the binding of the second antibody in the absenceof the first antibody. The alternative, where the binding of the firstantibody to the target is also detectably decreased in the presence ofthe second antibody, can, but need not be the case. That is, a secondantibody can inhibit the binding of a first antibody to the targetwithout that first antibody inhibiting the binding of the secondantibody to the target. However, where each antibody detectably inhibitsthe binding of the other antibody to its cognate epitope or ligand,whether to the same, greater, or lesser extent, the antibodies are saidto “cross-compete” with each other for binding of their respectiveepitope(s). Both competing and cross-competing antibodies areencompassed by the present invention. The term “competitor” antibody canbe applied to the first or second antibody as can be determined by oneof skill in the art. In some cases, the presence of the competitorantibody (e.g., the first antibody) reduces binding of the secondantibody to the target by at least 10%, e.g., at least any of 20%, 30%,40%, 50%, 60%, 70%, 80%, or more, e.g., so that binding of the secondantibody to target is undetectable in the presence of the first(competitor) antibody.

The terms “label,” “detectable moiety,” and like terms refer to acomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, chemical, or other physical means. For example, usefullabels include fluorescent dyes, luminescent agents, radioisotopes(e.g., ³²P, ³H), electron-dense reagents, enzymes (e.g., as commonlyused in an ELISA), biotin, digoxigenin, or haptens and proteins or otherentities which can be made detectable, e.g., by incorporating aradiolabel into a peptide or antibody specifically reactive with atarget analyte. Any method known in the art for conjugating an antibodyto the label may be employed, e.g., using methods described inHermanson, Bioconjugate Techniques 1996, Academic Press, Inc., SanDiego. The term “tag” can be used synonymously with the term “label,”but generally refers to an affinity-based moiety, e.g., a “His tag” forpurification, or a “strepavidin tag” that interacts with biotin.

A “labeled” molecule (e.g., nucleic acid, protein, or antibody) is onethat is bound, either covalently, through a linker or a chemical bond,or noncovalently, through ionic, van der Waals, electrostatic, orhydrogen bonds to a label such that the presence of the molecule may bedetected by detecting the presence of the label bound to the molecule.

The term “differentially expressed” or “differentially regulated” refersgenerally to a protein or nucleic acid biomarker that is overexpressed(upregulated) or underexpressed (downregulated) in one sample comparedto at least one other sample. In the context of the present disclosure,the term generally refers to overexpression of CLL-1 on a cancer cell(e.g., an AML cell or AML CSC) compared to a normal, non-cancer cell.

For example, the terms “overexpressed” or “upregulated” interchangeablyrefer to a protein or nucleic acid, generally a biomarker, that istranscribed or translated at a detectably greater than control level.The term includes overexpression due to transcription, posttranscriptional processing, translation, post-translational processing,cellular localization (e.g., organelle, cytoplasm, nucleus, cellsurface), and RNA and protein stability. Overexpression can be detectedusing conventional techniques for detecting biomarkers, whether mRNA(i.e., RT-PCR, hybridization) or protein (i.e., flow cytometry, imaging,ELISA, immunohistochemical techniques). Overexpression can be at leastany of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in comparisonto a normal cell.

The terms “agonist,” “activator,” “inducer” and like terms refer tomolecules that increase activity or expression as compared to a control.Agonists are agents that, e.g., bind to, stimulate, increase, activate,enhance activation, sensitize or upregulate the activity of the target.The expression or activity can be increased at least any of 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90% 100% or more than that in a control.In certain instances, the activation is any of 1.5-fold, 2-fold, 3-fold,4-fold, 5-fold, 10-fold, or more in comparison to a control.

The terms “inhibitor,” “repressor” or “antagonist” or “downregulator”interchangeably refer to a substance that results in a detectably lowerexpression or activity level as compared to a control. The inhibitedexpression or activity can be any of 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90% or less than that in a control. In certain instances, theinhibition is any of 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold,or more in comparison to a control.

A “control” sample or value refers to a sample that serves as areference, usually a known reference, for comparison to a test sample.For example, a test sample can be taken from a test condition, e.g., inthe presence of a test compound, and compared to samples from knownconditions, e.g., in the absence of the test compound (negativecontrol), or in the presence of a known compound (positive control). Inthe context of the present disclosure, an example of a negative controlwould be a biological sample from a known healthy (non-cancer)individual, and an example of a positive control would be a biologicalsample from a known AML patient. A control can also represent an averagevalue or a range gathered from a number of tests or results. One ofskill in the art will recognize that controls can be designed forassessment of any number of parameters. For example, a control can bedevised to compare therapeutic benefit based on pharmacological data(e.g., half-life) or therapeutic measures (e.g., comparison of benefitand/or side effects). Controls can be designed for in vitroapplications. One of skill in the art will understand which controls arevaluable in a given situation and be able to analyze data based oncomparisons to control values. Controls are also valuable fordetermining the significance of data. For example, if values for a givenparameter are widely variant in controls, variation in test samples willnot be considered as significant.

The term “diagnosis” refers to a relative probability that a subject hasa disorder such as cancer. Similarly, the term “prognosis” refers to arelative probability that a certain future outcome may occur in thesubject. For example, in the context of the present disclosure,prognosis can refer to the likelihood that an individual will developcancer, have recurrence, or the likely severity of the disease (e.g.,severity of symptoms, rate of functional decline, survival, etc.). Theterms are not intended to be absolute, as will be appreciated by any oneof skill in the field of medical diagnostics.

“Biopsy” or “biological sample from a patient” as used herein refers toa sample obtained from a patient having, or suspected of having, a CLL-1associated disorder. The sample can also be a blood sample or bloodfraction, e.g., white blood cell fraction, serum, or plasma. In someembodiments, the sample may be a tissue biopsy, such as needle biopsy,fine needle biopsy, surgical biopsy, etc. The sample can comprise atissue sample harboring a lesion or suspected lesion, although thebiological sample may be also be derived from another site, e.g., a siteof suspected metastasis, a lymph node, or from the blood. In some cases,the biological sample may also be from a region adjacent to the lesionor suspected lesion.

A “biological sample” can be obtained from a patient, e.g., a biopsy,from an animal, such as an animal model, or from cultured cells, e.g., acell line or cells removed from a patient and grown in culture forobservation. Biological samples include tissues and bodily fluids, e.g.,blood, blood fractions, lymph, saliva, urine, feces, etc.

The terms “therapy,” “treatment,” and “amelioration” refer to anyreduction in the severity of symptoms. In the case of treating cancer(e.g., AML), treatment can refer to, e.g., reducing tumor size, numberof cancer cells, growth rate, metastatic activity, reducing cell deathof non-cancer cells, reduced nausea and other chemotherapy orradiotherapy side effects, etc. The terms “treat” and “prevent” are notintended to be absolute terms. Treatment and prevention can refer to anydelay in onset, amelioration of symptoms, improvement in patientsurvival, increase in survival time or rate, etc. Treatment andprevention can be complete (undetectable levels of neoplastic cells) orpartial, such that fewer neoplastic cells are found in a patient thanwould have occurred without the present invention. The effect oftreatment can be compared to an individual or pool of individuals notreceiving the treatment, or to the same patient prior to treatment or ata different time during treatment. In some aspects, the severity ofdisease is reduced by at least 10%, as compared, e.g., to the individualbefore administration or to a control individual not undergoingtreatment. In some aspects the severity of disease is reduced by atleast 25%, 50%, 75%, 80%, or 90%, or in some cases, no longer detectableusing standard diagnostic techniques.

The terms “effective amount,” “effective dose,” “therapeuticallyeffective amount,” etc. refer to that amount of the therapeutic agentsufficient to ameliorate a disorder, as described above. For example,for the given parameter, a therapeutically effective amount will show anincrease or decrease of therapeutic effect at least any of 5%, 10%, 15%,20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%. Therapeuticefficacy can also be expressed as “-fold” increase or decrease. Forexample, a therapeutically effective amount can have at least any of a1.2-fold, 1.5-fold, 2-fold, 5-fold, or more effect over a control.

As used herein, the term “pharmaceutically acceptable” is usedsynonymously with physiologically acceptable and pharmacologicallyacceptable. A pharmaceutical composition will generally comprise agentsfor buffering and preservation in storage, and can include buffers andcarriers for appropriate delivery, depending on the route ofadministration.

The terms “dose” and “dosage” are used interchangeably herein. A doserefers to the amount of active ingredient given to an individual at eachadministration. For the present invention, the dose can refer to theconcentration of the antibody or associated components, e.g., the amountof therapeutic agent or dosage of radiolabel. The dose will varydepending on a number of factors, including frequency of administration;size and tolerance of the individual; severity of the condition; risk ofside effects; the route of administration; and the imaging modality ofthe detectable moiety (if present). One of skill in the art willrecognize that the dose can be modified depending on the above factorsor based on therapeutic progress. The term “dosage form” refers to theparticular format of the pharmaceutical, and depends on the route ofadministration. For example, a dosage form can be in a liquid, e.g., asaline solution for injection.

“Subject,” “patient,” “individual” and like terms are usedinterchangeably and refer to, except where indicated, mammals such ashumans and non-human primates, as well as rabbits, rats, mice, goats,pigs, and other mammalian species. The term does not necessarilyindicate that the subject has been diagnosed with a particular disease,but typically refers to an individual under medical supervision. Apatient can be an individual that is seeking treatment, monitoring,adjustment or modification of an existing therapeutic regimen, etc. A“cancer patient” or “AML patient” can refer to an individual that hasbeen diagnosed with cancer, is currently following a therapeuticregimen, or is at risk of recurrence, e.g., after surgery to remove atumor. In some embodiments, the cancer patient has been diagnosed withcancer and is a candidate for therapy. Cancer patients can includeindividuals that have not received treatment, are currently receivingtreatment, have had surgery, and those that have discontinued treatment.

In the context of treating cancer, a subject in need of treatment canrefer to an individual that has cancer or a pre-cancerous condition, hashad cancer and is at risk of recurrence, is suspected of having cancer,is undergoing standard treatment for cancer, such as radiotherapy orchemotherapy, etc.

“Cancer”, “tumor,” “transformed” and like terms include precancerous,neoplastic, transformed, and cancerous cells, and can refer to a solidtumor, or a non-solid cancer (see, e.g., Edge et al. AJCC Cancer StagingManual (7^(th) ed. 2009); Cibas and Ducatman Cytology: Diagnosticprinciples and clinical correlates (3^(rd) ed. 2009)). Cancer includesboth benign and malignant neoplasms (abnormal growth). “Transformation”refers to spontaneous or induced phenotypic changes, e.g.,immortalization of cells, morphological changes, aberrant cell growth,reduced contact inhibition and anchorage, and/or malignancy (see,Freshney, Culture of Animal Cells a Manual of Basic Technique (3^(rd)ed. 1994)). Although transformation can arise from infection with atransforming virus and incorporation of new genomic DNA, or uptake ofexogenous DNA, it can also arise spontaneously or following exposure toa carcinogen.

The term “cancer” can refer to leukemias, carcinomas, sarcomas,adenocarcinomas, lymphomas, solid and lymphoid cancers, etc. Examples ofdifferent types of cancer include, but are not limited to, acutemyelogenous leukemia (AML), chronic myelogenous leukemia (CML), B-celllymphoma, non-Hodgkin's lymphoma, Burkitt's lymphoma, Small Celllymphoma, Large Cell lymphoma, monocytic leukemia, myelogenous leukemia,acute lymphocytic leukemia, multiple myelomas, lung cancer (e.g.,non-small cell lung cancer or NSCLC), ovarian cancer, prostate cancer,colorectal cancer, liver cancer (i.e., hepatocarcinoma), renal cancer(i.e., renal cell carcinoma), bladder cancer, breast cancer, thyroidcancer, pleural cancer, pancreatic cancer, uterine cancer, cervicalcancer, testicular cancer, anal cancer, pancreatic cancer, bile ductcancer, gastrointestinal carcinoid tumors, esophageal cancer, gallbladder cancer, appendix cancer, small intestine cancer, stomach(gastric) cancer, cancer of the central nervous system, skin cancer,choriocarcinoma; head and neck cancer, osteogenic sarcoma, fibrosarcoma,neuroblastoma, glioma, and melanoma.

A “cancer target” or “cancer marker” is a molecule that isdifferentially expressed or processed in cancer, e.g., on a cancer cellor in the cancer milieu. Exemplary cancer targets are cell surfaceproteins such as CLL-1 (also, e.g., cell adhesion molecules andreceptors), intracellular receptors, hormones, and molecules such asproteases that are secreted by cells into the cancer milieu. Markers forspecific cancers are known in the art, e.g., CD45 for AML, CD34+CD38−for AML CSCs, MUC1 expression on colon and colorectal cancers, bombesinreceptors in lung cancer, and prostate specific membrane antigen (PSMA)on prostate cancer.

In some embodiments, the cancer target can be associated with a certaintype of cancer cell, e.g., AML, leukemia, myeloma, lymphoma, non-smallcell lung cancer cells, prostate cancer, colorectal cancer, breastcancer or ovarian cancer. A cell type specific target is typicallyexpressed at levels at least 2 fold greater in that cell type than in areference population of cells. In some embodiments, the cell typespecific marker is present at levels at least any of 3, 4, 5, 6, 7, 8,9, 10, 20, 50, 100, or 1000 fold higher than its average expression in areference population. Thus, the target can be detected or measured todistinguish the cell type or types of interest from other cells. Forexample, AML cancer targets include Ly86, LILRA1, and CD180.

A cancer stem cell (CSC) is a cell found in a tumor or blood cancer thatcan give rise to the cells that make up the bulk of the cancer. The CSCcan also be self-renewing, similar to a normal (non-cancer) stem cell.CSCs can thus mediate metastasis by migrating to a non-tumor tissue inan individual and starting a “new” tumor. CSCs make up a very smallpercentage of any given cancer, depending on the stage that the canceris detected. For example, the average frequency of CSCs in a sample ofAML cells is believed to be about 1:10,000. Hematopoietic CSCs can beidentified as CD34+, similar to normal hematopoietic stem cells (HSCs).

The terms “internalize,” “internalization,” “endocytose,” “endocytosis,”“engulf,” and like terms refer to uptake of a substance by a cell, e.g.,by antibody (or receptor)-mediated endocytosis or phagocytosis. Theresults of the ADC assays in Example 5 indicate that the presentlydisclosed CLL-1 antibodies can be internalized.

The terms “engraft” or “engraftment” refers to the ability of a cell tosurvive, proliferate, and/or properly localize upon introduction into anindividual or tissue. In the case of a cancer stem cell (CSC), the termcan refer to the ability of the CSC to generate a tumor de novo or tospread to a different site. The term is commonly used to describe theability of a population of cells to survive and function in a xenograftmodel (e.g., engraftment of human cells in a mouse). Engraftment ofhematopoietic cells can be determined as described, e.g., inWO2006/047569. Engraftment of tumor cells can be determined asdescribed, e.g., in Beckhove et al. (2003) Int. J. Cancer 105:444.

The term “nucleic acid” refers to deoxyribonucleotides orribonucleotides and polymers thereof in either single- ordouble-stranded form, and complements thereof. The term “polynucleotide”refers to a linear sequence of nucleotides. The term “nucleotide”typically refers to a single unit of a polynucleotide, i.e., a monomer.Nucleotides can be naturally occurring ribonucleotides ordeoxyribonucleotides, or synthetic or modified versions thereof.Examples of polynucleotides contemplated herein include single anddouble stranded DNA, single and double stranded RNA (including siRNA),and hybrid molecules having mixtures of single and double stranded DNAand RNA.

The words “complementary” or “complementarity” refer to the ability of anucleic acid in a polynucleotide to form a base pair with anothernucleic acid in a second polynucleotide. For example, the sequence A-G-Tis complementary to the sequence T-C-A. Complementarity may be partial,in which only some of the nucleic acids match according to base pairing,or complete, where all the nucleic acids match according to basepairing.

A variety of methods of specific DNA and RNA measurements that usenucleic acid hybridization techniques are known to those of skill in theart (see, Sambrook, Id.). Some methods involve electrophoreticseparation (e.g., Southern blot for detecting DNA, and Northern blot fordetecting RNA), but measurement of DNA and RNA can also be carried outin the absence of electrophoretic separation (e.g., quantitative PCR,dot blot, or array).

The words “protein”, “peptide”, and “polypeptide” are usedinterchangeably to denote an amino acid polymer or a set of two or moreinteracting or bound amino acid polymers. The terms apply to amino acidpolymers in which one or more amino acid residue is an artificialchemical mimetic of a corresponding naturally occurring amino acid, aswell as to naturally occurring amino acid polymers, those containingmodified residues, and non-naturally occurring amino acid polymer.

The term “amino acid” refers to naturally occurring amino acids,modified or synthetic amino acids, as well as amino acid analogs andamino acid mimetics that function similarly to naturally occurring aminoacids. Naturally occurring amino acids are those encoded by the geneticcode. Modified amino acids include, e.g., hydroxyproline,γ-carboxyglutamate, and 0-phosphoserine. Amino acid analogs refers tocompounds that have the same basic chemical structure as a naturallyoccurring amino acid, e.g., an a carbon that is bound to a hydrogen, acarboxyl group, an amino group, and an R group, e.g., homoserine,norleucine, methionine sulfoxide, methionine methyl sulfonium. Suchanalogs may have modified R groups (e.g., norleucine) or modifiedpeptide backbones, but retain the same basic chemical structure as anaturally occurring amino acid. Amino acid mimetics refers to chemicalcompounds that have a structure that is different from the generalchemical structure of an amino acid, but that functions similarly to anaturally occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical or associated, e.g., naturallycontiguous, sequences. Because of the degeneracy of the genetic code, alarge number of functionally identical nucleic acids encode mostproteins. For instance, the codons GCA, GCC, GCG and GCU all encode theamino acid alanine. Thus, at every position where an alanine isspecified by a codon, the codon can be altered to another of thecorresponding codons described without altering the encoded polypeptide.Such nucleic acid variations are “silent variations,” which are onespecies of conservatively modified variations. Every nucleic acidsequence herein which encodes a polypeptide also describes silentvariations of the nucleic acid. One of skill will recognize that incertain contexts each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, silent variations of a nucleic acidwhich encodes a polypeptide are implicit in a described sequence withrespect to the expression product, but not with respect to actual probesequences.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention. The following amino acids aretypically conservative substitutions for one another: 1) Alanine (A),Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N),Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine(L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C),Methionine (M) (see, e.g., Creighton, Proteins (1984)).

The terms “identical” or “percent identity,” in the context of two ormore nucleic acids, or two or more polypeptides, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of nucleotides, or amino acids, that are the same (i.e.,about 60% identity, e.g., at least any of 65%, 70%, 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over aspecified region, when compared and aligned for maximum correspondenceover a comparison window or designated region) as measured using a BLASTor BLAST 2.0 sequence comparison algorithms with default parameters, orby manual alignment and visual inspection. See e.g., the NCBI web siteat ncbi.nlm.nih.gov/BLAST. Such sequences are then said to be“substantially identical.” Percent identity is typically determined overoptimally aligned sequences, so that the definition applies to sequencesthat have deletions and/or additions, as well as those that havesubstitutions. The algorithms commonly used in the art account for gapsand the like. Typically, identity exists over a region comprising anantibody epitope, or a sequence that is at least about 25 amino acids ornucleotides in length, or over a region that is 50-100 amino acids ornucleotides in length, or over the entire length of the referencesequence.

The term “recombinant” when used with reference, e.g., to a cell, ornucleic acid, protein, or vector, indicates that the cell, nucleic acid,protein or vector, has been modified by the introduction of aheterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, for example, recombinant cells express genes that arenot found within the native (non-recombinant) form of the cell orexpress native genes that are otherwise abnormally expressed, underexpressed or not expressed at all.

The term “heterologous,” with reference to a polynucleotide orpolypeptide, indicates that the polynucleotide or polypeptide comprisestwo or more subsequences that are not found in the same relationship toeach other in nature. For instance, a heterologous polynucleotide orpolypeptide is typically recombinantly produced, having two or moresequences from unrelated genes arranged to make a new functional unit,e.g., a promoter from one source and a coding region from anothersource. Similarly, a heterologous protein indicates that the proteincomprises two or more subsequences that are not found in the samerelationship to each other in nature (e.g., a fusion protein).

III. CLL-1 Associated Disorders

The presently described antibodies can be used to detect and treat CLL-1associated disorders, i.e., diseases correlated with elevated or reducedcell surface expression of CLL-1 as compared to CLL-1 expression in astandard control (e.g., a normal, non-disease, non-cancer cell). CLL-1expression is normally limited to myeloid lineage cells, e.g., dendriticcells, granulocytes, and monocytes in the peripheral blood and spleen.Elevated CLL-1 levels are associated with cancer, in particular, inhematopoietic CSCs (e.g., LSCs), and in myeloproliferative disorders,including leukemias such as AML (acute myelogenous or myeloproliferativeleukemia), MDS (myelodysplastic syndrome), myelofibrosis, CMML (chronicmyelomonocytic leukemia), multiple myeloma, plasmacytoma, and CIVIL(chronic myelogenous or myeloproliferative leukemia). See Bakker et al.(2004) Cancer Res. 64:8443; Van Rhenen et al. (2007) Blood 110:2659-66;Zhao et al. (2010) Haematologica (2010) 95:71; Van Rhenen et al. (2007)Leukemia 21:1700; and Herrmann et al. (2012) Haematologica 97:219.

AML cells can be characterized and distinguished from other cells bydetecting cell surface marker expression. Aside from being CLL-1+, AMLcells can be CD33+(though some are CD33-), CD45+, and CDw52+. AML blasts(including LSCs) are typically CD34+CD38-. HSCs and LSCs can becharacterized by expression of CD34, but the former do not expressCLL-1. MDS cells can be characterized by expression of CDS, CD7, CD13,and CD34. CIVIL cells can be characterized by expression of 7-ADD, CD33,CD34, and CD38.

Myelodysplastic Syndromes (MDS) include a group of closely-related bloodformation disorders, in which the bone marrow shows qualitative andquantitative changes suggestive of a preleukemic process, but having achronic course that does not necessarily terminate as acute leukemia. Avariety of terms, including preleukemia, refractory anemia, refractorydysmyelopoietic anemia, smoldering or subacute leukemia, dysmyelopoieticsyndrome (DMPS), and myelodysplasia, have all been used to describe MDS.These conditions are all characterized by a cellular marrow withimpaired maturation (dysmyelopoiesis) and a reduction in the number ofblood cells. DMPS is characterized by presence of megablastoids,megarkaryocyte dysplasia, and an increase in number of abnormal blastcells, reflective of enhanced granulocyte maturation process. Patientswith DMPS show chromosomal abonormalities similar to those found inacute myeloid leukemia and progress to acute myeloid leukemia in acertain fraction of afflicted patients.

Chronic myeloproliferative disorders are a collection of conditionscharacterized by increased number of mature and immature granulocytes,erythrocytes, and platelets. Chronic myeloproliferative disorders cantransition to other forms within this group, with a tendency toterminate in acute myeloid leukemia. Specific diseases within this groupinclude polycythemia vera, chronic myeloid leukemia, agnogenic myeloidleukemia, essential thrombocythemia, and chronic neutrophilic leukemia.

Myelofibrosis is characterized by scarring of the bone marrow thatresults in reduced number of red and white blood cells, and platelets.Myelofibrotic scarring can result from leukemia, but can have othercauses, such as thrombocytosis or adverse drug effects.

IV. CLL-1 Antibodies

Provided herein are CLL-1 antibodies (i.e., CLL-1 specific antibodies,anti-CLL-1) that specifically bind to human CLL-1, in particular to theextracellular domain of a CLL-1 expressing cell. In some embodiments,the CLL-1 antibodies bind an epitope that includes a component that isoutside the C lectin domain such that the antibodies bind a polypeptideconsisting of the C lectin domain with lower affinity that a polypeptideconsisting of the C lectin and stalk domains of CLL-1, or theextracellular domain of CLL-1. In some embodiments, the CLL-1 antibodybinds a polypeptide consisting of the C-lectin domain of CLL-1 with a Kdat least 5-fold higher than a polypeptide consisting of the C-lectin andstalk domains of CLL-1 (e.g., any of 10, 20, 50, 100 or higher fold).For example, the CLL-1 antibodies designated as M26 and M31 bind aminoacids 101-265 of human CLL-1 with higher affinity than amino acids141-265 of human CLL-1 (with reference to SEQ ID NO:2). In someembodiments, the CLL-1 antibody binds the C lectin domain with a Kd thatis at least 5, 10, 20, 50, or 100-fold higher than full length CLL-1 (orthe full length extracellular domain of CLL-1).

In some embodiments, the CLL-1 antibodies have an affinity for humanCLL-1 with a Kd of 1000 pM or lower, e.g., any of 800 pM, 700 pM, 600pM, 500 pM, 400 pM, 300 pM, 200 pM, 100 pM, or lower. In someembodiments, the CLL-1 antibodies have an affinity for human CLL-1 witha Kd of 10 nM or lower, e.g., 1 nM or lower, 1-10 nM, 100-1000 pM,10-1000 pM, about 1 nM or lower, 1-500 pM. In some embodiments, theCLL-1 antibodies also bind to primate CLL-1, e.g., cynomolgus CLL-1,with Kd that is 10 nM, 1 nM, 500 pM or less. In some embodiments, theCLL-1 antibodies bind cynomolgus CLL-1 with a Kd that is within an orderof magnitude of the Kd for human CLL-1. One of skill will understandthat lower Kd values indicate higher affinity.

In some embodiments, the CCL-1 antibodies bind a broad range of CLL-1glycosylation variants. In some embodiments, the CLL-1 antibodies bind aform (e.g., a glycosylation variant) of CLL-1 that is expressed on AMLcells. For example, the presently described CLL-1 antibodies can bind atleast any of 65, 70, 75, 80, 85, 90, 95 or higher percent of the cellsin an AML cell culture (e.g., HL60, THP1, and U937 cell lines). In someembodiments, the CLL-1 antibodies can bind at least any of 50, 60, 65,70, 75, 80, 85, 90, 95 or higher percent of the cells in an AML patentsample (e.g., a PBMC sample or biopsy from an AML patient). One of skillwill understand that, in such a cell binding assay, that an appropriateconcentration of antibody is added, e.g., so that there are sufficientantibody molecules present to bind the number of cells in the culture orsample.

Surprisingly, CLL-1 antibodies described herein can inhibit growth ofCLL-1-expressing cells in vitro and in vivo even in the absence of aconjugated cytotoxic agent. Given the high percentage of binding to AMLcells from patient samples, the presently described antibodies provide auseful therapeutic option for AML patients, as well as those sufferingfrom CLL-1+ MDS or CML.

The CLL-1 antibodies described herein also show complement dependentcytotoxicity (CDC) activity (see, e.g., FIGS. 1A-1C) and antibody drugconjugate (ADC) activity (see Example 5). These CLL-1 antibodies canalso thus be used to target CLL-1 expressing cells for destruction, e.g.in the absence of a conjugated cytotoxic agent.

CLL-1 antibodies described herein have unique cell binding activitiescompared to previously characterized antibodies. For example, thepresently described antibodies bind an epitope that is present on ahigher percentage of primary cells from AML patients. These antibodiescan be used for detecting cancer cells that display an epitope that istargeted with high affinity by at least one of the CLL-1 antibodiesdisclosed herein. In some embodiments, those cancer cells can then betargeted for destruction with the same CLL-1 antibody. Such methods caninclude treating an individual having CLL-1 expressing cancer cellscomprising administering the CLL-1 antibody to the individual.

In some embodiments, the invention includes CLL-1 antibodies thatcompete for binding to CLL-1 with a competitor antibody selected fromthe group consisting of:

-   -   an antibody having the CDR sequences of M26 (see Example 1,        Table 3)    -   an antibody having the CDR sequences of M31;    -   an antibody having the CDR sequences of G4;    -   an antibody having the CDR sequences of M22;    -   an antibody having the CDR sequences of M29;    -   an antibody having the CDR sequences of M2;    -   an antibody having the CDR sequences of M5; and    -   an antibody having the CDR sequences of G12.

In some embodiments, the CLL-1 antibodies competes for binding to CLL-1with an antibody selected from the group consisting of:

-   -   an antibody comprising variable region sequences with        substantial identity (at least (85, 90, 95, or 98% identity) to        those of M26 (Vh=SEQ ID NO:4; V1=SEQ ID NO:6)    -   an antibody comprising variable region sequences with        substantial identity to those of M31 (Vh=SEQ ID NO:8; V1=SEQ ID        NO:10);    -   an antibody comprising variable region sequences with        substantial identity to those of G4 (Vh=SEQ ID NO:12; V1=SEQ ID        NO:14);    -   an antibody comprising variable region sequences with        substantial identity to those of M22 (Vh=SEQ ID NO:16; V1=SEQ ID        NO:18);    -   an antibody comprising variable region sequences with        substantial identity to those of M29 (Vh=SEQ ID NO:20; V1=SEQ ID        NO:22);    -   an antibody comprising variable region sequences with        substantial identity to those of M2 (Vh=SEQ ID NO:24; V1=SEQ ID        NO:26);    -   an antibody comprising variable region sequences with        substantial identity to those of M5 (Vh=SEQ ID NO:28; V1=SEQ ID        NO:30); and    -   an antibody comprising variable region sequences with        substantial identity to those of G12 (Vh=SEQ ID NO:32; V1=SEQ ID        NO:34). In some embodiments, the substantially identical        antibody has the same heavy and light chain CDR sequences as the        original antibody.

Numerous types of competitive binding assays are known, including solidphase direct or indirect radioimmunoassay (RIA); solid phase direct orindirect enzyme immunoassay (EIA), sandwich competition assay (seeStahli et al., Methods in Enzymology 9:242-253 (1983)); solid phasedirect biotin-avidin EIA (see Kirkland et al., J. Immunol. 137:3614-3619(1986)); solid phase direct labeled assay; solid phase direct labeledsandwich assay (see Harlow and Lane, Antibodies, A Laboratory Manual,Cold Spring Harbor Press (1988)); solid phase direct label RIA usingI-125 label (see Morel et al., Molec. Immunol. 25(1):7-15 (1988)); solidphase direct biotin-avidin EIA (Cheung et al., Virology 176:546-552(1990)); and direct labeled RIA (Moldenhauer et al., Scand. J. Immunol.32:77-82 (1990)). Typically, such an assay involves the use of purifiedantigen bound to a solid surface or cells bearing either of these, anunlabelled test immunoglobulin and a labeled reference immunoglobulin.Competitive inhibition is measured by determining the amount of labelbound to the solid surface or cells in the presence of the testimmunoglobulin. Usually the test immunoglobulin is present in excess.Antibodies identified by competition assay (competing antibodies)include antibodies binding to the same epitope as the reference antibodyand antibodies binding to an adjacent epitope sufficiently proximal tothe epitope bound by the reference antibody for steric hindrance tooccur. Usually, when a competing antibody is present in excess, it willinhibit specific binding of a reference antibody to a common antigen byat least 50 or 75%.

In some embodiments, the CLL-1 antibody binds the same epitope as anantibody selected from the group consisting of:

-   -   an antibody having the CDR sequences of M26 (see Example 1,        Table 3)    -   an antibody having the CDR sequences of M31;    -   an antibody having the CDR sequences of G4;    -   an antibody having the CDR sequences of M22;    -   an antibody having the CDR sequences of M29;    -   an antibody having the CDR sequences of M2;    -   an antibody having the CDR sequences of M5; and    -   an antibody having the CDR sequences of G12.

In some embodiments, the CLL-1 antibody has light chain CDR sequencesand heavy chain CDR sequences having up to 1, 2, or 3 amino acidsubstitutions, additions, or deletions/CDR relative to the CDR sequencesof an antibody selected from the group consisting of M26, M31, G4, M22,M29, M2, M5, and G12. In some embodiments, the light chain CDR sequencesinclude up to 1, 2, or 3 amino acid substitutions, additions ordeletions/CDR relative to the light chain CDR sequences of theaforementioned CLL-1 antibodies. In some embodiments, the heavy chainCDR sequences include up to 1, 2, or 3 amino acid substitutions,additions, or deletions/CDR relative to the heavy chain CDR sequences ofthe aforementioned CLL-1 antibodies. In some embodiments, substitution,addition or deletion occurs in only 1, 2, 3, 4, or 5 CDRs of the 6 totalCDRs.

In some embodiments, the CLL-1 antibody is selected from the groupconsisting of:

-   -   an antibody having the CDR sequences of M26 (see Example 1,        Table 3);    -   an antibody having the CDR sequences of M31;    -   an antibody having the CDR sequences of G4;    -   an antibody having the CDR sequences of M22;    -   an antibody having the CDR sequences of M29;    -   an antibody having the CDR sequences of M2;    -   an antibody having the CDR sequences of M5; and    -   an antibody having the CDR sequences of G12. In some        embodiments, any one or more of the CDR sequences includes 1, 2,        or 3 conservative amino acid substitutions compared to the        original antibody CDR sequences.

In some embodiments, the CLL-1 antibody is selected from the groupconsisting of:

-   -   an antibody comprising variable region sequences with        substantial identity (at least (85, 90, 95, or 98% identity) to        those of M26 (Vh=SEQ ID NO:4; V1=SEQ ID NO:6)    -   an antibody comprising variable region sequences with        substantial identity to those of M31 (Vh=SEQ ID NO:8; V1=SEQ ID        NO:10);    -   an antibody comprising variable region sequences with        substantial identity to those of G4 (Vh=SEQ ID NO:12; V1=SEQ ID        NO:14);    -   an antibody comprising variable region sequences with        substantial identity to those of M22 (Vh=SEQ ID NO:16; V1=SEQ ID        NO:18);    -   an antibody comprising variable region sequences with        substantial identity to those of M29 (Vh=SEQ ID NO:20; V1=SEQ ID        NO:22);    -   an antibody comprising variable region sequences with        substantial identity to those of M2 (Vh=SEQ ID NO:24; V1=SEQ ID        NO:26);    -   an antibody comprising variable region sequences with        substantial identity to those of M5 (Vh=SEQ ID NO:28; V1=SEQ ID        NO:30); and    -   an antibody comprising variable region sequences with        substantial identity to those of G12 (Vh=SEQ ID NO:32; V1=SEQ ID        NO:34).

In some embodiments, the antibody also has at least one activityselected from

-   -   Binding to human CLL-1 with a Kd of 10 nM or lower, e.g., 1 nM        or lower, 1-10 nM, 100-1000 pM, 10-1000 pM, about 1 nM or lower,        1-500 pM, etc.;    -   An EC50 of 200 ng/ml or less in a CDC assay with HL60 cells or        CLL-1 expressing AML cells from an AML patient;    -   An EC50 of 100 pM of less in a ADC assay with HL60 cells or        CLL-1 expressing AML cells from an AML patient; and    -   Reducing cell growth of CLL-1-expressing cells (e.g., HL60, AML        cells), compared to cell growth in the absence of the antibody.

Any of the antibodies described herein can be a chimeric antibody or ahumanized antibody. In some embodiments, the antibody is a CLL-1-bindingantibody fragment, e.g., an Fab. In some embodiments, the CLL-1 antibodyis labeled with a detectable agent, e.g., as described below. In someembodiments, the CLL-1 antibody is attached to a therapeutic agent,e.g., a chemotherapeutic or cytotoxic agent as described below.

A. Methods of Making Antibodies

For preparation of the presently described antibodies, e.g.,recombinant, monoclonal, or polyclonal antibodies, many techniques knownin the art can be used (see, e.g., Kohler & Milstein, Nature 256:495-497(1975); Kozbor et al., Immunology Today 4: 72 (1983); Cole et al., pp.77-96 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc.(1985); Coligan, Current Protocols in Immunology (1991); Harlow & Lane,Antibodies, A Laboratory Manual (1988); and Goding, MonoclonalAntibodies: Principles and Practice (2d ed. 1986)). The genes encodingthe heavy and light chains of an antibody of interest can be cloned froma cell, e.g., the genes encoding a monoclonal antibody can be clonedfrom a hybridoma and used to produce a recombinant monoclonal antibody.Gene libraries encoding heavy and light chains of monoclonal antibodiescan also be made from hybridoma or plasma cells. Random combinations ofthe heavy and light chain gene products generate a large pool ofantibodies with different antigenic specificity (see, e.g., Kuby,Immunology (3^(rd) ed. 1997)). Techniques for the production of singlechain antibodies or recombinant antibodies (U.S. Pat. No. 4,946,778,U.S. Pat. No. 4,816,567) can be adapted to produce antibodies topolypeptides of this invention. Also, transgenic mice, or otherorganisms such as other mammals, can be used to express humanized orhuman antibodies (see, e.g., U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, Marks et al., Bio/Technology10:779-783 (1992); Lonberg et al., Nature 368:856-859 (1994); Morrison,Nature 368:812-13 (1994); Fishwild et al., Nature Biotechnology14:845-51 (1996); Neuberger, Nature Biotechnology 14:826 (1996); andLonberg & Huszar, Intern. Rev. Immunol. 13:65-93 (1995)). Alternatively,phage display technology can be used to identify antibodies andheteromeric Fab fragments that specifically bind to selected antigens(see, e.g., McCafferty et al., Nature 348:552-554 (1990); Marks et al.,Biotechnology 10:779-783 (1992)). Antibodies can also be madebispecific, i.e., able to recognize two different antigens (see, e.g.,WO 93/08829, Traunecker et al., EMBO J. 10:3655-3659 (1991); and Sureshet al., Methods in Enzymology 121:210 (1986)). Antibodies can also beheteroconjugates, e.g., two covalently joined antibodies, orimmunotoxins (see, e.g., U.S. Pat. No. 4,676,980, WO 91/00360; WO92/200373; and EP 03089).

Antibodies can be produced using any number of expression systems,including prokaryotic and eukaryotic expression systems. In someembodiments, the expression system is a mammalian cell expression, suchas a hybridoma, or a CHO cell expression system. Many such systems arewidely available from commercial suppliers. In embodiments in which anantibody comprises both a V_(H) and V_(L) region, the V_(H) and V_(L)regions may be expressed using a single vector, e.g., in a di-cistronicexpression unit, or under the control of different promoters. In otherembodiments, the V_(H) and V_(L) region may be expressed using separatevectors. A V_(H) or V_(L) region as described herein may optionallycomprise a methionine at the N-terminus.

An antibody of the invention can also be produced in various formats,including as a Fab, a Fab′, a F(ab′)2, a scFv, or a dAB. The antibodyfragments can be obtained by a variety of methods, including, digestionof an intact antibody with an enzyme, such as pepsin (to generate(Fab′)₂ fragments) or papain (to generate Fab fragments); or de novosynthesis. Antibody fragments can also be synthesized using recombinantDNA methodology. In some embodiments, the CLL-1 antibody comprisesF(ab′)₂ fragments that specifically bind CLL-1. An antibody of theinvention can also include a human constant region. See, e.g.,Fundamental Immunology (Paul ed., 4d ed. 1999); Bird, et al., Science242:423 (1988); and Huston, et al., Proc. Natl. Acad. Sci. USA 85:5879(1988).

Methods for humanizing non-human antibodies (i.e., using CDRs fromnon-human antibodies) are also known in the art. Generally, a humanizedantibody has one or more amino acid residues from a source which isnon-human. These non-human amino acid residues are often referred to asimport residues, which are typically taken from an import variabledomain. Humanization can be essentially performed following the methodof Winter and co-workers (see, e.g., Jones et al., Nature 321:522-525(1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al.,Science 239:1534-1536 (1988) and Presta, Curr. Op. Struct. Biol.2:593-596 (1992)), by substituting rodent CDRs or CDR sequences for thecorresponding sequences of a human antibody. Such humanized antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

In some cases, the antibody or antibody fragment can be conjugated toanother molecule, e.g., polyethylene glycol (PEGylation) or serumalbumin, to provide an extended half-life in vivo. Examples ofPEGylation of antibody fragments are provided in Knight et al. Platelets15:409, 2004 (for abciximab); Pedley et al., Br. J. Cancer 70:1126, 1994(for an anti-CEA antibody); Chapman et al., Nature Biotech. 17:780,1999; and Humphreys, et al., Protein Eng. Des. 20: 227, 2007). Theantibody or antibody fragment can also be labeled, or conjugated to atherapeutic agent as described below.

B. Binding Affinity

The specificity of the binding can be defined in terms of thecomparative dissociation constants (Kd) of the antibody (or othertargeting moiety) for target, as compared to the dissociation constantwith respect to the antibody and other materials in the environment orunrelated molecules in general. Typically, the Kd for the antibody withrespect to the unrelated material will be at least 2-fold, 3-fold,4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold or higherthan Kd with respect to the target.

The desired affinity for an antibody, e.g., high (pM to low nM), medium(low nM to 100 nM), or low (about 100 nM or higher), may differdepending upon whether it is being used as a diagnostic or therapeutic.Without being limited to theory, in one example, an antibody with mediumaffinity may be more successful in localizing to a tumor as compared toone with a high affinity. Thus, antibodies having different affinitiescan be used for diagnostic and therapeutic applications.

A targeting moiety will typically bind with a Kd of less than about 1000nM, e.g., less than 250, 100, 50, 20 or lower nM. In some embodiments,the Kd of the affinity agent is less than 15, 10, 5, or 1 nM. In someembodiments, the Kd is 1-100 nM, 0.1-50 nM, 0.1-10 nM, or 1-20 nM. Thevalue of the dissociation constant (Kd) can be determined by well-knownmethods, and can be computed even for complex mixtures by methods asdisclosed, e.g., in Caceci et al., Byte (1984) 9:340-362.

Affinity of an antibody, or any targeting agent, for a target can bedetermined according to methods known in the art, e.g., as reviewed inErnst et al. Determination of Equilibrium Dissociation Constants,Therapeutic Monoclonal Antibodies (Wiley & Sons ed. 2009).

Quantitative ELISA, and similar array-based affinity methods can beused. ELISA (Enzyme linked immunosorbent signaling assay) is anantibody-based method. In some cases, an antibody specific for target ofinterest is affixed to a substrate, and contacted with a samplesuspected of containing the target. The surface is then washed to removeunbound substances. Target binding can be detected in a variety of ways,e.g., using a second step with a labeled antibody, direct labeling ofthe target, or labeling of the primary antibody with a label that isdetectable upon antigen binding. In some cases, the antigen is affixedto the substrate (e.g., using a substrate with high affinity forproteins, or a Strepavidin-biotin interaction) and detected using alabeled antibody (or other targeting moiety). Several permutations ofthe original ELISA methods have been developed and are known in the art(see Lequin (2005) Clin. Chem. 51:2415-18 for a review).

The Kd, Kon, and Koff can also be determined using surface plasmonresonance (SPR), e.g., as measured by using a Biacore T100 system. SPRtechniques are reviewed, e.g., in Hahnfeld et al. Determination ofKinetic Data Using SPR Biosensors, Molecular Diagnosis of InfectiousDiseases (2004). In a typical SPR experiment, one interactant (target ortargeting agent) is immobilized on an SPR-active, gold-coated glassslide in a flow cell, and a sample containing the other interactant isintroduced to flow across the surface. When light of a given frequencyis shined on the surface, the changes to the optical reflectivity of thegold indicate binding, and the kinetics of binding.

Binding affinity can also be determined by anchoring a biotinylatedinteractant to a streptaviden (SA) sensor chip. The other interactant isthen contacted with the chip and detected, e.g., as described inAbdessamad et al. (2002) Nuc. Acids Res. 30:e45.

C. Determining CLL-1 Epitope

The site of antibody binding to CLL-1 can be mapped using knowntechniques for epitope mapping. One of skill will appreciate that theapproach used for epitope mapping can vary depending on the antigen,e.g., where it is expressed in the cell, post-translationalmodifications of the primary polypeptide sequence, and differencesbetween antigen structure on different cells or in differentenvironments.

CLL-1 is a transmembrane protein with approximately 200 extracellularamino acids. The extracellular domain is glycosylated, and includes a Clectin domain. The epitope for a CLL-1 antibody can be determined orpartially determined by varying the primary sequence or glycosylationstate of CLL-1, and comparing the affinity of the CLL-1 antibody to thedifferent variants of CLL-1.

Such epitope mapping can be carried out in vitro, e.g., by screeningphage display libraries or synthetic peptide libraries, e.g., usingbeads or other solid matrices. Linear epitopes are typically about sixamino acids, though this can vary somewhat. In order to mimic linearepitopes present in a protein, synthetic peptides can be madecorresponding to the sequence. In some embodiments, this sequence isextended on the N and/or C terminals to provide additional amino acidresidues that are present in the flanking sequences in the protein. Thiscan more closely mimic the primary, and to a certain extent, thesecondary structure environment of the epitope. Additionally, residuesincluding but not limited to one or more glycines or gamma amino butyricacid, can be appended to either terminus to provide a spacer to minimizesteric interactions with, for example, a solid phase used in animmunoassay. Spacer length is often varied to determine empirically thebest structure.

Because of the variable nature of the epitope and the potential effectsdue to the flanking sequences, in some embodiments, one can use peptidesthat vary in length by extending the N or C terminals by a certainnumber of residues. Another approach utilizes repeating peptideepitopes, or alternating epitopes with intervening spacer residues. Thelength of these peptides is often varied according to the number ofrepeating units desired.

One approach for epitope mapping is to synthesize overlapping peptides,for example 20 residues in length, with a six residue overlap, whichcover the primary sequence of the CLL-1 extracellular region. If suchpeptide screening is used to map the epitope, peptides can be modifiedto overcome the undesirable interactions with solid phase supports usedin immunoassays. One way is to substitute hydrophobic residues in thepeptide with hydrophilic ones, in order to reduce or minimize thehydrophobic interactions, and increased peptide accessibility.Similarly, charged peptide residues can be substituted with nonchargedresidues to eliminate ionic interactions with the solid phase. Peptidescan also be modified by adding spacer groups of a variety of structuresto position the peptide epitope further from the solid phase andminimize steric hindrance.

Peptides can be synthesized to reflect post-translational modificationsthat are present on the native protein, or the native protein ontargeted cells. Modifications include but are not limited toglycosylation and phosphorylation at specific sites in the protein.

Another approach for determining the epitope is to express CLL-1variants in cells, and compare CLL-1 antibody affinities between thedifferent variants. The CLL-1 variants can be designed as described forthe peptide studies. In addition, glycosylated residues (e.g.,asparagine, arginine, serine, threonine, tyrosine) can be substituted todetermine whether the epitope includes a glycosylation site. Similarly,phosphorylated residues (serine, threonine, tyrosine) can besubstituted.

The epitope can also be determined or partially determined by comparingantibody affinity for different types of CLL-1 expressing cells. Forexample, antibody affinity can be determined and compared for primaryAML cells, e.g., AML blasts and engrafted AML tumor cells; for AML celllines, for other non-cancerous myeloid cells, etc.

D. CDC, ADCC, and ADC Assays

The presently described antibodies are effective for Cell dependentcytotoxicity (CDC), Antibody dependent cell-mediated cytotoxicity(ADCC), and Antibody drug conjugate cytotoxicity (ADC) of cells thatexpress CLL-1. Exemplary cells that express CLL-1 include cell linesthat express heterologous, recombinant CLL-1 (e.g., human CLL-1); humanAML cell lines such as HL60, THP1, TF1-alpha, U937, and OCI AML-5 (thefirst four of which are available from ATCC); primary cells from one ormore AML patients (e.g., PBMC or engrafted tumor cells); human CML celllines such as K562 and KU812 (available from ATCC); and primary cellsfrom one or more CIVIL or MDS patients.

An antibody is described as having CDC activity and mediating CDC if itresults in complement dependent killing of cells that express theantibody target. CDC assays are known in the art, and are described,e.g., in Gazzano-Santoro et al. (1997) J. Immunol. Methods 202:163;Idusogie et al. (2000) 1 Immunol. 164:4178; and in Example 6 below. CDCkits and services are commercially available, e.g. from GeneScript® andCell Technology Inc.

In brief, the assay is typically carried out in vitro, and includesantibody binding to a cell expressing the antibody target on itssurface. Complement components, including Clq which binds to the Chregion of the antibody, are added. The complement components theninteract to kill the targeted cell. CDC is measured after a period ofincubation of generally between 4 and 24 hours, for example, bydetermining the release of intracellular enzyme or granules known to bepresent in the targeted cell, by comparing the starting and endingtarget cell population, etc.

An antibody is described as having ADCC activity and mediating ADCC ifit results in killing of antibody-bound cells (e.g., CLL-1 expressingcells) by effector cells. Effector cells are typically natural killercells, but can also be macrophages, neutrophils, or eosinophils.Genetically engineered effector cell lines have also been developed foruse in ADCC assays (see, e.g., Schnueriger et al. (2011) Mol. Immunol.48:1512). ADCC assays are known in the art, and are described, e.g., inPerussia and Loza (2000) Methods in Mol. Biol. 121:179; Bretaudeau andBonnaudet (2011) BMC Proceedings 5(Suppl 8):P63; and in Example 12below. ADCC kits and services are commercially available, e.g. fromGeneScript® and Promega®.

In brief, the assay is typically carried out in vitro, and includesantibody binding to a cell expressing the antibody target on itssurface. Effector cells are added that recognize antibody-bound cells,typically through an Fc receptor such as CD16. The effector cells killthe antibody-bound cell, e.g., by releasing cytotoxins that causeapoptosis. Cell death is detected by release of a detectable elementwithin the target cells (e.g., Cr51) or by detection of an elementinvolved in the cell mediated toxicity (e.g., activation of NFATsignaling in effector cells).

An antibody is described as having antibody-drug conjugate (ADC)activity (or mediating ADC) if the antibody, when conjugated with acytotoxic agent (drug), results in killing (inhibiting survival) a cellthat expresses the target of the antibody, in this case, CLL-1.Appropriate cytotoxic agents are known in the art, e.g., saporin,doxorubicin, daunomycin, vinca-alkaloids, taxoids, tubulin agents (e.g.,Maytansin, auristatin), and DNA agents (e.g., calicheamicin,duocarmycin, pyrrolobenzodiazepine dimers), etc. ADC assays are known inthe art, e.g., as described in Gerber et al. (2009) 3:247, and in theExamples below.

E. Internalization

The CLL-1 antibodies described herein can be internalized intoCLL-1-expressing cells, including CLL-1 AML cells. That is, a CLL-1expressing cell can internalize the antibodies described herein. TheCLL-1 antibodies described herein provide an effective means fortargeting such cells, e.g., with detectable or cytotoxic conjugates.

The percent internalization and internalization rate of an antibody canbe evaluated by using methods known in the art, including, e.g., flowcytometry (FACS) and confocal fluorescent microscopy. Such methods aredescribed, e.g., in Lue et al. (2007) Nature Protocols (Nature Med.13:587-96); Cho et al. (2010) Biomacromolecules and Corbani et al.(2004) Endocrinology 145:2876-85, and as described herein.

For FACS and confocal microscopy, cells are incubated with afluorescently-labeled targeting agent, e.g., antibody. The cells aretypically selected to express the target of the labeled antibody, e.g.,CLL-1. Control cells can then be used that do not express the target.Internalization typically occurs at 37° C., but not at 4° C., whichprovides another control for the reaction. The cells can thus becontacted with the labeled agent and incubated at 37° C. or 4° C. (e.g.,to detect binding without internalization).

Unbound, and surface-bound agent is removed by washing the cells, e.g.,in an acid wash, followed by wash with a buffer at normal pH.

If adherent cells are used, the cells are removed from substrate priorto flow cytometry. The percentage of fluorescent cells indicates thepercent internalization of the fluorescently-labeled agent. Percentinternalization can also be expressed, e.g., as a percent of initiallabeled agent added to the cells.

Internalization of an agent can also be evaluated by determining thelocalization of the fluorescently labeled agent by confocal microscopy.Methods of using confocal microscopy to determine internalization aredescribed in, e.g., Xiao et al. (2008) Chem. Eur. 1, 14:1769-1775.Briefly, the cells are contacted with labeled agent and incubated asdescribed above. Following incubation, the cells can be incubated onice, washed in PBS buffer at 4° C., treated with 0.25% trypsin (toremove from substrate, if applicable). The cell suspension can then beapplied to slides for confocal fluorescent microscopy. Suitable confocalmicroscopes include the FV500-IX81 confocal microscope (Olympus AmericaInc.; Center Valley, Pa.) and Eclipse Ti-E (Nikon Instruments Inc.;Melville, N.Y.).

V. Diagnostic Applications

The CLL-1 antibodies described herein specifically bind CLL-1-expressingcells. CLL-1 antibodies can thus be used for in vitro and in vivodiagnostic assays to detect CLL-1-expressing cells (e.g., AML cells andAML CSCs). For example, a sample (e.g., blood sample or tissue biopsy)can be obtained from a patient and contacted with a CLL-1 antibody, andthe presence of a CLL-1-expressing cell in the patient sample can bedetermined by detecting antibody binding. Antibody binding can bedetected directly (e.g., where the antibody itself is labeled) or byusing a second detection agent, such as a secondary antibody. Thedetectable label can be associated with an antibody of the invention,either directly, or indirectly, e.g., via a chelator or linker.

In some embodiments, the CLL-1 antibody is contacted with a biologicalsample from an individual having or suspected of having a CLL-1associated disorder, and antibody binding to a cell in the sample isdetermined, wherein higher or lower than normal antibody bindingindicates that the individual has a CLL-1 associated disorder. In someembodiments, the biological sample is a blood sample or blood fraction(e.g., serum, plasma, platelets, red blood cells, white blood cells,PBMCs). In some embodiments, the biological sample is a tissue sample(biopsy), e.g., from a suspected tumor site, or from a tissue that isknown to be affected, e.g., to determine the boundaries of a knowntumor.

Biopsies are typically performed to obtain samples from tissues, i.e.,non-fluid cell types. The biopsy technique applied will depend on thetissue type to be evaluated (e.g., breast, skin, colon, prostate,kidney, lung, bladder, lymph node, liver, bone marrow, airway or lung).In the case of a cancer the technique will also depend on the size andtype of the tumor (e.g., solid, suspended, or blood), among otherfactors. Representative biopsy techniques include, but are not limitedto, excisional biopsy, incisional biopsy, needle biopsy, surgicalbiopsy, and bone marrow biopsy. An “excisional biopsy” refers to theremoval of an entire tumor mass with a small margin of normal tissuesurrounding it. An “incisional biopsy” refers to the removal of a wedgeof tissue that includes a cross-sectional diameter of the tumor. Adiagnosis or prognosis made by endoscopy or fluoroscopy can require a“core-needle biopsy” of the tumor mass, or a “fine-needle aspirationbiopsy” which generally obtains a suspension of cells from within thetumor mass. Biopsy techniques are discussed, for example, in Harrison'sPrinciples of Internal Medicine, Kasper, et al., eds., 16th ed., 2005,Chapter 70, and throughout Part V.

Any method of detecting antibody binding to a cell in a sample can beused for the present diagnostic assays. Methods of detecting antibodybinding are well known in the art, e.g., flow cytometry, fluorescentmicroscopy, ELISAs, etc. In some embodiments, the method comprisespreparing the biological sample for detection prior to the determiningstep. For example, a subpopulation of cells (e.g., white blood cells,CD34+ cells, CD45+ cells, etc.) can be separated from the rest of thesample from the individual (e.g., other blood components) or cells in atissue can be suspended for easier detection.

In some embodiments, the percentage of CLL-1-expressing cells in thesample is determined and compared to a control, e.g., a sample from anindividual or group of individuals that are known to have a CLL-1associated disorder (positive control) or from an individual or group ofindividuals that are known not to have a CLL-1 associated disorder(normal, healthy, non-disease, or negative control). In someembodiments, the control is a standard range of CLL-1 expressionestablished for a given tissue. A higher or lower than normal percentageof CLL-1 expressing cells, or higher or lower expression level,indicates that the individual has a CLL-1 associated disorder.

In some embodiments, a labeled CLL-1 antibody can be provided(administered) to an individual to determine the applicability of anintended therapy. For example, a labeled antibody may be used to detectCLL-1 density within a diseased area, where the density is typicallyhigh relative to non-diseased tissue. A labeled antibody can alsoindicate that the diseased area is accessible for therapy. Patients canthus be selected for therapy based on imaging results. Anatomicalcharacterization, such as determining the precise boundaries of acancer, can be accomplished using standard imaging techniques (e.g., CTscanning, MM, PET scanning, etc.).

In some embodiments, labeled CLL-1 antibodies as described herein can befurther associated with a therapeutic compound, e.g., to form a“theranostic” composition. For example, an CLL-1 antibody can be linked(directly or indirectly) to both a detectable label and a therapeuticagent, e.g., a cytotoxic agent to kill CLL-1-expressing cancer cells. Insome embodiments, a labeled CLL-1 antibody is used for diagnosis and/orlocalization of a CLL-1 expressing cancer cell, and the CLL-1 expressingcancer cell is then targeted with a separate therapeutic CLL-1 specificantibody. In some embodiments, the diagnostic CLL-1 specific antibody isone that is not internalized into CLL-1-expressing cells at a high rateor percentage.

In some embodiments, the therapeutic CLL-1 antibody is internalized intoCLL-1-expressing cells at a high rate or percentage.

A. Labels

A diagnostic agent comprising a CLL-1 antibody can include anydiagnostic agent known in the art, as provided, for example, in thefollowing references: Armstrong et al., Diagnostic Imaging, 5^(th) Ed.,Blackwell Publishing (2004); Torchilin, V. P., Ed., Targeted Delivery ofImaging Agents, CRC Press (1995); Vallabhajosula, S., Molecular Imaging:Radiopharmaceuticals for PET and SPECT, Springer (2009). A diagnosticagent can be detected by a variety of ways, including as an agentproviding and/or enhancing a detectable signal. Detectable signalsinclude, but are not limited to, gamma-emitting, radioactive, echogenic,optical, fluorescent, absorptive, magnetic, or tomography signals.Techniques for imaging the diagnostic agent can include, but are notlimited to, single photon emission computed tomography (SPECT), magneticresonance imaging (MRI), optical imaging, positron emission tomography(PET), computed tomography (CT), x-ray imaging, gamma ray imaging, andthe like. The terms “detectable agent,” “detectable moiety,” “label,”“imaging agent,” and like terms are used synonymously herein.

In some embodiments, the label can include optical agents such asfluorescent agents, phosphorescent agents, chemiluminescent agents, andthe like. Numerous agents (e.g., dyes, probes, labels, or indicators)are known in the art and can be used in the present invention. (See,e.g., Invitrogen, The Handbook—A Guide to Fluorescent Probes andLabeling Technologies, Tenth Edition (2005)). Fluorescent agents caninclude a variety of organic and/or inorganic small molecules or avariety of fluorescent proteins and derivatives thereof. For example,fluorescent agents can include but are not limited to cyanines,phthalocyanines, porphyrins, indocyanines, rhodamines, phenoxazines,phenylxanthenes, phenothiazines, phenoselenazines, fluoresceins,benzoporphyrins, squaraines, dipyrrolo pyrimidones, tetracenes,quinolines, pyrazines, corrins, croconiums, acridones, phenanthridines,rhodamines, acridines, anthraquinones, chalcogenopyrylium analogues,chlorins, naphthalocyanines, methine dyes, indolenium dyes, azocompounds, azulenes, azaazulenes, triphenyl methane dyes, indoles,benzoindoles, indocarbocyanines, benzoindocarbocyanines, and BODIPY™derivatives. Fluorescent dyes are discussed, for example, in U.S. Pat.No. 4,452,720, U.S. Pat. No. 5,227,487, and U.S. Pat. No. 5,543,295.

The label can also be a radioisotope, e.g., radionuclides that emitgamma rays, positrons, beta and alpha particles, and X-rays. Suitableradionuclides include but are not limited to ²²⁵Ac, ⁷²As, ²¹¹At, ¹¹B,¹²⁸Ba, ²¹²Bi, ⁷⁵Br, ⁷⁷Br, ¹⁴C, ¹⁰⁹Cd, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ¹⁸F, ⁶⁷Ga, ⁶⁸Ga,³H, ¹⁶⁶Ho, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³⁰I, ¹³¹I, ¹⁷⁷Lb, ¹³N, ¹⁵O, ³²P, ³³P,²¹²Pb, ¹⁰³Pd, ¹⁸⁶Re ¹⁸⁸Re, ⁴⁷Sc, ¹⁵³Sm, ⁸⁹Sr, ^(99m)Tc, ⁸⁸Y and ⁹⁰Y. Insome embodiments, radioactive agents can include ¹¹¹In-DTPA,^(99m)Tc(CO)₃-DTPA, ^(99m)Tc(CO)₃-ENPy2, ^(62/64/67)Cu-TETA,^(99m)Tc(CO)₃-IDA, and ^(99m)Tc(CO)₃triamines (cyclic or linear). Insome embodiments, the agents can include DOTA and its various analogswith ¹¹¹In, ¹⁷⁷Lu, ¹⁵³Sm, ^(88/90)Y, ^(62/64/67)Cu, or ⁶⁷¹⁶⁸Ga. In someembodiments, a nanoparticle can be labeled by incorporation of lipidsattached to chelates, such as DTPA-lipid, as provided in the followingreferences: Phillips et al., Wiley Interdisciplinary Reviews:Nanomedicine and Nanobiotechnology, 1(1): 69-83 (2008); Torchilin, V. P.& Weissig, V., Eds. Liposomes 2nd Ed.: Oxford Univ. Press (2003);Elbayoumi, T. A. & Torchilin, V. P., Eur. J. Nucl. Med. Mol. Imaging33:1196-1205 (2006); Mougin-Degraef, M. et al., Int'l J. Pharmaceutics344:110-117 (2007).

In some embodiments, the diagnostic agent can be associated with asecondary binding ligand or to an enzyme (an enzyme tag) that willgenerate a colored product upon contact with a chromogenic substrate.Examples of suitable enzymes include urease, alkaline phosphatase,(horseradish) hydrogen peroxidase and glucose oxidase. Secondary bindingligands include, e.g., biotin and avidin or streptavidin compounds asknown in the art.

In some embodiments, the labeled antibody can be further associated to acomposition that improves stability in vivo, e.g. PEG or a nanoparticlesuch as a liposome, as described in more detail below.

B. Methods of Labeling

Techniques for conjugating detectable and therapeutic agents toantibodies are well known (see, e.g., Arnon et al., “MonoclonalAntibodies For Immunotargeting Of Drugs In Cancer Therapy”, inMonoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp.243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., “Antibodies For DrugDelivery” in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.),pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers OfCytotoxic Agents In Cancer Therapy: A Review” in Monoclonal Antibodies'84: Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); and Thorpe et al., “The Preparation And CytotoxicProperties Of Antibody-Toxin Conjugates”, Immunol. Rev., 62:119-58(1982)).

Typically, the antibody is attached to detectable moiety in an area thatdoes not interfere with binding to the epitope. Thus in some cases, thedetectable moiety is attached to the constant region, or outside theCDRs in the variable region. One of skill in the art will recognize thatthe detectable moiety can be located elsewhere on the antibody, and theposition of the detectable moiety can be adjusted accordingly. In someembodiments, the ability of the antibody to associate with the epitopeis compared before and after attachment to the detectable moiety toensure that the attachment does not unduly disrupt binding.

In some embodiments, the antibody can be associated with an additionaltargeting moiety. For example, an antibody fragment, peptide, or aptamerthat binds a different site on the target molecule or target cell can beconjugated to the antibody to optimize target binding, e.g., to a cancercell.

VI. Therapeutic Applications

CLL-1-expressing cells such as AML cells can be targeted using the CLL-1antibodies described herein. CLL-1 expression is elevated on AML cellsand CSCs (e.g., AML CSCs). CLL-1 is not significantly expressed onnormal CD34+ hematopoietic stem cells (HSCs), thus CSCs can bedistinguished from HSCs using the present CLL-1 antibodies. Highaffinity CLL-1 antibodies that recognize a CLL-1 epitope common to AMLcells, and thus able to universally bind to AML cells, is particularlyvaluable, as AML has a very high rate of recurrence. As noted above, atherapeutic composition comprising CLL-1 antibody can further include adetectable label to form a theranostic composition, e.g., for detectionand localization of CLL-1 expressing cells, and monitoring oftherapeutic effect.

As demonstrated herein, the present CLL-1 antibodies can inhibit cancercell growth (proliferation and/or engraftment) and thus can beconsidered chemotherapeutic agents alone. The following disclosureprovides examples of chemotherapeutic and cytotoxic agents that can belinked to CLL-1 antibody for additional effect on CLL-1-expressingcells.

A chemotherapeutic (anti-cancer) agent can be any agent capable ofreducing cancer growth, interfering with cancer cell replication,directly or indirectly killing cancer cells, reducing metastasis,reducing tumor blood supply, etc. Chemotherapeutic agents thus includecytotoxic agents. Cytotoxic agents include but are not limited tosaporin, taxanes, vinca alkaloids, anthracycline, and platinum-basedagents. Classes of chemotherapeutic agents include but are not limitedto alkylating agents, antimetabolites, e.g, methotrexate, plantalkaloids, e.g., vincristine, and antibiotics, e.g., doxorubicin as wellas miscellaneous drugs that do not fall in to a particular class such ashydroxyurea. Platinum-based drugs, exemplified by cisplatin andoxaliplatin, represent a major class of chemotherapeutics. These drugsbind to DNA and interfere with replication. Taxanes, exemplified bytaxol, represent another major class of chemotherapeutics. Thesecompounds act by interfering with cytoskeletal and spindle formation toinhibit cell division, and thereby prevent growth of rapidly dividingcancer cells. Other chemotherapeutic drugs include hormonal therapy.

More than one therapeutic agent can be combined, either in the samecomposition, or in separate compositions. The therapeutic agent(s) canalso be combined with additional therapeutic agents as appropriate forthe particular individual. Common therapeutic agents provided to cancerpatients include medications to address pain, nausea, anemia, infection,inflammation, and other symptoms commonly experienced by cancerpatients.

Antibodies can be attached to a therapeutic agent, detectable agent, ornanocarrier using a variety of known cross-linking agents. Methods forcovalent or non-covalent attachment of polypeptides are well known inthe art. Such methods may include, but are not limited to, use ofchemical cross-linkers, photoactivated cross-linkers and/or bifunctionalcross-linking reagents. Exemplary methods for cross-linking moleculesare disclosed in U.S. Pat. No. 5,603,872 and U.S. Pat. No. 5,401,511.Non-limiting examples of cross-linking reagents include glutaraldehyde,bifunctional oxirane, ethylene glycol diglycidyl ether, carbodiimidessuch as 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide ordicyclohexylcarbodiimide, bisimidates, dinitrobenzene,N-hydroxysuccinimide ester of suberic acid, disuccinimidyl tartarate,dimethyl-3,3′-dithio-bispropionimidate, azidoglyoxal,N-succinimidyl-3-(2-pyridyldithio)propionate and4-(bromoadminoethyl)-2-nitrophenylazide.

In some embodiments, the CLL-1 antibody is associated with ananocarrier. For antibodies conjugated to nanocarriers (e.g.,liposomes), a certain number of antibodies will be present on thesurface, i.e., at a given surface density. In some embodiments, thenanocarrier will have at least 5 antibodies per nanocarrier, e.g., atleast 10, 30, 40, 50, 75, 100 or higher antibodies per nanocarrier. Oneof skill in the art will understand that surface density represents anaverage range, as the number of antibodies per nanocarrier will not beabsolutely uniform for all members of the population.

Nanocarriers include vesicles such as liposomes and micelles, as well aspolymeric nanoparticles, etc. Nanocarriers are useful for delivery oftherapeutic and diagnostic agents, but can be particularly useful forshielding cytotoxic agents used to treat cancer. The nanocarrier cancomprise lipids (e.g., phospholipids), hydrophilic polymers, hydrophobicpolymers, amphipatic compounds, cross-linked polymers, and a polymericmatrix (see, e.g., WO2009/110939). Depending on the application, thenanocarrier can be designed to have a particular size, half-life, shelflife, and leakage rate.

Preparation of nanocarriers, such as an antibody targeted liposome,polymeric nanoparticle, or extended shelf-life liposome, is described,e.g., in U.S. Pat. Nos. 6,465,188, 7,122,202, 7,462,603 and 7,550,441.

In some embodiments, the antibody is linked to a stabilizing moiety suchas PEG, or a liposome or other nanocarrier. U.S. Pat. Nos. 4,732,863 and7,892,554 and Chattopadhyay et al. (2010) Mol Pharm 7:2194 describemethods for attaching the selected antibody to PEG, PEG derivatives, andnanoparticles (e.g., liposomes). Liposomes containingphosphatidyl-ethanolamine (PE) can be prepared by established proceduresas described herein. The inclusion of PE provides an active functionalsite on the liposomal surface for attachment.

The antibody conjugate can also be formulated to provide more than oneactive compound, e.g., additional chemotherapeutic or cytotoxic agents,cytokines, or growth inhibitory agents. The active ingredients may alsoprepared as sustained-release preparations (e.g., semi-permeablematrices of solid hydrophobic polymers (e.g., polyesters, hydrogels (forexample, poly (2-hydroxyethyl-methacrylate), or poly (vinylalcohol)),polylactides. The antibodies and immunocongugates can be entrapped in ananoparticle prepared, for example, by coacervation techniques or byinterfacial polymerization, for example, hydroxymethylcellulose orgelatin microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nanoparticles andnanocapsules) or in macroemulsions.

The CLL-1 antibodies described herein can kill CLL-1-expressing cellsalone, or in combination with a cytotoxic agent. In some embodiments,the method of treatment comprises administering to an individual aneffective amount of a therapeutic CLL-1 antibody or CLL-1 antibodyconjugate, e.g., a CLL-1 antibody attached to a therapeutic agent. Insome embodiments, the individual has been diagnosed with cancer, e.g.,AML. In some embodiments, the individual is receiving or has receivedcancer therapy, e.g., surgery, radiotherapy, or chemotherapy. In someembodiments, the individual has been diagnosed, but the cancer is inremission.

In some embodiments, the method further comprises monitoring theindividual for progression of the cancer. In some embodiments, the doseof the CLL-1 antibody or CLL-1 antibody conjugate for eachadministration is determined based on the therapeutic progress of theindividual, e.g., where a higher dose of chemotherapeutic isadministered if the individual is not responding sufficiently totherapy.

In some embodiments, the invention can include an antibody orantibody-targeted composition and a physiologically (i.e.,pharmaceutically) acceptable carrier. The term “carrier” refers to atypically inert substance used as a diluent or vehicle for a diagnosticor therapeutic agent. The term also encompasses a typically inertsubstance that imparts cohesive qualities to the composition.Physiologically acceptable carriers can be liquid, e.g., physiologicalsaline, phosphate buffer, normal buffered saline (135-150 mM NaCl),water, buffered water, 0.4% saline, 0.3% glycine, glycoproteins toprovide enhanced stability (e.g., albumin, lipoprotein, globulin, etc.),and the like. Since physiologically acceptable carriers are determinedin part by the particular composition being administered as well as bythe particular method used to administer the composition, there are awide variety of suitable formulations of pharmaceutical compositions ofthe present invention (See, e.g., Remington's Pharmaceutical Sciences,17^(th) ed., 1989).

The compositions of the present invention may be sterilized byconventional, well-known sterilization techniques or may be producedunder sterile conditions. Aqueous solutions can be packaged for use orfiltered under aseptic conditions and lyophilized, the lyophilizedpreparation being combined with a sterile aqueous solution prior toadministration. The compositions can contain pharmaceutically acceptableauxiliary substances as required to approximate physiologicalconditions, such as pH adjusting and buffering agents, tonicityadjusting agents, wetting agents, and the like, e.g., sodium acetate,sodium lactate, sodium chloride, potassium chloride, calcium chloride,sorbitan monolaurate, and triethanolamine oleate. Sugars can also beincluded for stabilizing the compositions, such as a stabilizer forlyophilized antibody compositions.

Dosage forms can be prepared for mucosal (e.g., nasal, sublingual,vaginal, buccal, or rectal), parenteral (e.g., subcutaneous,intravenous, intramuscular, or intraarterial injection, either bolus orinfusion), oral, or transdermal administration to a patient. Examples ofdosage forms include, but are not limited to: dispersions;suppositories; ointments; cataplasms (poultices); pastes; powders;dressings; creams; plasters; solutions; patches; aerosols (e.g., nasalsprays or inhalers); gels; liquid dosage forms suitable for oral ormucosal administration to a patient, including suspensions (e.g.,aqueous or non-aqueous liquid suspensions, oil-in-water emulsions, or awater-in-oil liquid emulsions), solutions, and elixirs; liquid dosageforms suitable for parenteral administration to a patient; and sterilesolids (e.g., crystalline or amorphous solids) that can be reconstitutedto provide liquid dosage forms suitable for parenteral administration toa patient.

Injectable (e.g., intravenous) compositions can comprise a solution ofthe antibody or antibody-targeted composition suspended in an acceptablecarrier, such as an aqueous carrier. Any of a variety of aqueouscarriers can be used, e.g., water, buffered water, 0.4% saline, 0.9%isotonic saline, 0.3% glycine, 5% dextrose, and the like, and mayinclude glycoproteins for enhanced stability, such as albumin,lipoprotein, globulin, etc. Often, normal buffered saline (135-150 mMNaCl) will be used. The compositions can contain pharmaceuticallyacceptable auxiliary substances to approximate physiological conditions,such as pH adjusting and buffering agents, tonicity adjusting agents,wetting agents, e.g., sodium acetate, sodium lactate, sodium chloride,potassium chloride, calcium chloride, sorbitan monolaurate,triethanolamine oleate, etc.

In some embodiments, the antibody-targeted composition can be formulatedin a kit for intravenous administration.

Formulations suitable for parenteral administration, such as, forexample, by intraarticular (in the joints), intravenous, intramuscular,intratumoral, intradermal, intraperitoneal, and subcutaneous routes,include aqueous and non-aqueous, isotonic sterile injection solutions,which can contain antioxidants, buffers, bacteriostats, and solutes thatrender the formulation isotonic with the blood of the intendedrecipient, and aqueous and non-aqueous sterile suspensions that caninclude suspending agents, solubilizers, thickening agents, stabilizers,and preservatives.

The pharmaceutical preparation can be packaged or prepared in unitdosage form. In such form, the preparation is subdivided into unit dosescontaining appropriate quantities of the active component, e.g.,according to the dose of the therapeutic agent or concentration ofantibody. The unit dosage form can be a packaged preparation, thepackage containing discrete quantities of preparation, in unit-dose ormulti-dose sealed containers, such as ampoules and vials. Thecomposition can, if desired, also contain other compatible therapeuticagents.

The antibody (or antibody-targeted composition) can be administered byinjection or infusion through any suitable route including but notlimited to intravenous, subcutaneous, intramuscular or intraperitonealroutes. An example of administration of a pharmaceutical compositionincludes storing the antibody at 10 mg/ml in sterile isotonic aqueoussaline solution for injection at 4° C., and diluting it in either 100 mlor 200 ml 0.9% sodium chloride for injection prior to administration tothe patient. The antibody is administered by intravenous infusion overthe course of 1 hour at a dose of between 0.2 and 10 mg/kg. In otherembodiments, the antibody is administered by intravenous infusion over aperiod of between 15 minutes and 2 hours. In still other embodiments,the administration procedure is via sub-cutaneous bolus injection.

The dose of antibody is chosen in order to provide effective therapy forthe patient and is in the range of less than 0.1 mg/kg body weight toabout 25 mg/kg body weight or in the range 1 mg-2 g per patient. In somecases, the dose is in the range 1-100 mg/kg, or approximately 50 mg-8000mg/patient. The dose may be repeated at an appropriate frequency whichmay be in the range once per day to once every three months, dependingon the pharmacokinetics of the antibody (e.g., half-life of the antibodyin the circulation) and the pharmacodynamic response (e.g., the durationof the therapeutic effect of the antibody). In some embodiments, the invivo half-life of between about 7 and about 25 days and antibody dosingis repeated between once per week and once every 3 months.

Administration can be periodic. Depending on the route ofadministration, the dose can be administered, e.g., once every 1, 3, 5,7, 10, 14, 21, or 28 days or longer (e.g., once every 2, 3, 4, or 6months). In some cases, administration is more frequent, e.g., 2 or 3times per day. The patient can be monitored to adjust the dosage andfrequency of administration depending on therapeutic progress and anyadverse side effects, as will be recognized by one of skill in the art.

Thus in some embodiments, additional administration is dependent onpatient progress, e.g., the patient is monitored betweenadministrations. For example, after the first administration or round ofadministrations, the patient can be monitored for rate of tumor growth,recurrence (e.g., in the case of a post-surgical patient), or generaldisease-related symptoms such as weakness, pain, nausea, etc.

For the treatment of cancer, an antibody or antibody-targetedcomposition (e.g., including a therapeutic and/or diagnostic agent) canbe administered at the initial dosage of about 0.001 mg/kg to about 1000mg/kg daily and adjusted over time. A daily dose range of about 0.01mg/kg to about 500 mg/kg, or about 0.1 mg/kg to about 200 mg/kg, orabout 1 mg/kg to about 100 mg/kg, about 5 to about 10 mg/kg, or about 10mg/kg to about 50 mg/kg, can be used. The in vivo xenograft resultsdescribed herein indicate that a dose between 5-20 mg antibody/kg bodyweight is effective for dramatic reduction of tumor growth.

The dosage is varied depending upon the requirements of the patient, theseverity of the condition being treated, and the targeted compositionbeing employed. For example, dosages can be empirically determinedconsidering the type and stage of cancer diagnosed in a particularpatient. The dose administered to a patient, in the context of thepresent invention, should be sufficient to affect a beneficialtherapeutic response in the patient over time. The size of the dose willalso be determined by the existence, nature, and extent of any adverseside-effects that accompany the administration of a particular targetedcomposition in a particular patient, as will be recognized by theskilled practitioner.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentireties for all purposes.

VII. EXAMPLES A. Example 1: Characterization of CLL-1 Antibody Sequencesand Structure

Human CLL-1 was used to generate antibodies in mice. Antibodies specificfor CLL-1 were selected and cloned into hybridomas for stable productionof monoclonal antibodies. A number of antibodies specific for CLL-1 werecloned and characterized for sequence and antibody structure. These dataare shown in Tables 1-3 below. The heavy and light chain variable regionsequences are shown in the sequence listing.

TABLE 1 Antibody structures Clone Isotype VH DH JH VK JK M26 IgG2bVhJ558.b14 PseudoD3 JH4 IGKV9-124*01 JK2 M31 IgG2a VhJ558.b14 DSP2.2 JH2IGKV3-10*01 JK1 G4 IgG1 VHJ558 DSP2.2 JH4 IGKV10-96*01 JK1 M22 IgG2aIGHV1-61*01 DSP2.5 JH4 IGKV8-19*01 JK5 M29 IgG1 VhJ558.b14 DSP2.2 JH2IGKV19-93*01 JK1 M2 IgG1 IGHV1-36*01 DSP2.9 JH4 IGKV9-124*01 JK2 M5IgG2a 14-1-39 DQ52a.1 JH2 IGKV8-30*01 JK1 G12 IgG1 DFL16.3 JH1IGKV3-10*01 JK2

TABLE 2 J sequences Clone J HC J LC M26 CTRDDGYYGYAMDYW CLQYAIYPYTF M31CARPIYFDNDYFDYW CQQNNYDPWTF G4 CARTDDYDDYTMDYW CQQGKTLLWTF M22CAIYYGNPSYYAMDYW CQNDYSYPFTF M29 CARYYDYDYYFDYW CLQYDYLWTF M2CTRDDGYYDYAMDYW CLQYASYPYTF M5 CTLTGRFDYW CQQYYSYRTF G12 CARVYNWHFDVWCQQNNEDPYTFJ HCs: SEQ ID NOs: 35-42 (top to bottom)

J LCs: SEQ ID Nos: 43-50

TABLE 3 CDR sequences Clone CDR H1 CDR H2 CDR H3 CDR L1 CDR L2 CDR L3M26 GYTFTSYF INPYNDGS TRDDGYYGYAMDY QELSGY AAS LQYAIYPYT M31 GYTFTSYVINPYNDGT ARPIYFDNDYFDY ESVDSYGNSF LAS QQNNYDPWT G4 GYSFTGYT INPYNDGTARTDDYDDYTMDY HDISNY YTS QQGKTLLWT M22 GYTFTRYW IDPSDTET AIYYGNPSYYAMDYQNLLNSGNQKKY WAS QNDYSYPFT M29 GYIFTSYV INPYNDGT ARYYDYDYYFDY QDINKY YTSLQYDYLWT M2 GYTFTSYF INPYNDGT TRDDGYYDYAMDY QEISVY AAS LQYASYPYT M5GFNIKDDY IDPEKGDT TLTGRFDY QSLLYSSNQKNN WAS QQYYSYRT G12 GYTFPSSNIYPGNGDT ARVYNWHFDV ESVDGYGDIF FAS QQNNEDPYT

CDR H1s: SEQ ID NOs: 51-58 CDR H2s: SEQ ID NOs: 59-66 CDR H3s: SEQ IDNOs: 67-74 CDR L1s: SEQ ID NOs: 75-82 CDR L2s: SEQ ID NOs: 83-90 CDRL3s: SEQ ID NOs: 91-98 B. Example 2: Epitope Binding Studies

For certain clones, epitope mapping was carried out, and compared to thelocation of binding to CLL-1 for known antibodies. These antibodiesinclude Nuvelo/X1057 (US20100285037), Crucell/X357 (U.S. Pat. No.7,741,443), and Goat anti CLL-1. A summary is shown in Table 4 below.CLL-1 or the C lectin domain of CLL-1 was expressed in 293T cells.Non-transfected 293T cells, or 293T cells transfected with mouse CLL-1were used as controls.

TABLE 4 Epitope binding 293T 293T C Clone 293T 293T mCLL-1 hCLL-1 lectindomain M13 − − + + M26 − − + − M31 − − + − X357 − − + + X1057 − − + +Goat anti-CLL-1 − − + +

The data show that clones M26 and M31 bind to human CLL-1, but that theC lectin domain is not sufficient for significant binding.

The M26 and M31 antibodies were also tested for binding to Cynomolgusmonkey CLL-1. These animals can be used for clinical studies, thus it isuseful to have target-specific antibodies that bind the Cynomolgusspecies homolog of a human antibody target. M26 was found to bindCynomolgus CLL-1 with high affinity.

Additional Cynomolgus CLL-1 binding studies were carried out usingELISA. The results are shown in Table 5 below.

TABLE 5 Cynomolgus CLL-1 binding Clone Relative binding M26 ++++ M31 ++G4 ++++ M2 +++ M5 +++ M13 − M22 − M29 + M41 +

C. Example 3: Affinity Testing

Affinity testing was carried out for the CLL-1 antibody clones. Briefly,biotinylated CLL-1 (25 ug/ml) is loaded onto strepavidin sensor tips for2 hours at 22 C. Ab-Ag dissociation curves were generated at threedifferent concentrations for each antibody (10, 30, and 90 ug/ml) usinga global 1:1 curve fitting. The results are shown in Table 6 below.

TABLE 6 Affinity Kd (pM) Clone Affinity for hCLL-1 M26 214 M31 611 G4 53M2 205 M5 553 M13 854 M22 388 M29 1480 M41 387 D11 436 E3 447 G2 4640 G61492 G8 980 G10 194 G12 70 G14 187 G16 2357 G23 543 G26 134 G30 241

D. Example 4: Binding to AML Cell Lines and AML Patient Samples

The CLL-1 antibodies were tested for binding to recombinant 293 cellsexpressing human CLL-1, and two AML, cell lines, HL60 and OCI AML-5. Thepercentage of live cells with antibody binding, as detected by FACS, isshown in Table 7 below.

TABLE 7 Antibody binding to cell lines (%) Clone 293 CLL-1 HL60 OCIAML-5 M26 99.9 91 92.7 M31 76 91 89.7 G4 83.2 83.2 M2 99.9 90.3 96.3 M51.1 1.7 2.3 M13 90 13.6 35.3 M22 97 37.2 57.6 M29 99.9 84.6 87.6 M41 9995.2 87.1 B10 99.9 92 16 D11 82 E3 99.9 86 8 G2 99.9 10 88.8 G6 99.983.7 G8 98 65.5 G10 99.9 88.5 G12 99.9 88.3 G14 99.9 86.2 G16 56.4 G2381.4 G26 99.9 92.3 G30 99.9 89.2

Previously characterized CLL-1 antibodies typically bind primary AMLcells with high variability, which is problematic for broad use withpatient samples. Some do not detectably bind samples from certainpatients. The presently disclosed antibodies were tested for binding toprimary cells from AML patient samples by FACS. Two groups of sampleswere studied: the first consisting of 6 patients, the other consistingof a larger cohort of 37. Each antibody clone was not tested for bindingto every sample in the groups. Results of binding are shown in Table 8.M26 and M31 were further found to bind 90% or more cells from AMLpatient primary cell samples by FACS.

TABLE 8 Binding of primary AML samples (Positive/Total number of patientsamples tested) Clone Group 1 Group 2 M26 6/6 32/37 M31 2/3  5/12 G4 2/24/6 M2 2/2 M5 1/6  0/20 M13 2/3  0/20 M22 3/5  1/35 M29  4/26 M41 6/6B10 5/6 D11 E3 4/6 2/6 G2 1/2 G6 1/2 G8 2/2 G10 1/1 2/5 G12 2/2 G14 2/2G16 2/2 G23 2/2 G26 2/2 G30 1/1

E. Example 5: Antibody-Drug Conjugate (ADC) Assays

Antibody-Drug Conjugate (ADC) assays were carried out on AML cell linesHL60 and OCI AML-5, as well as recombinant 293 cells expressing CLL-1.Briefly, cells were incubated with various concentrations ofsaporin-conjugated antibodies for 48-72 hours at 37 C. Cell viabilitywas determined by DHL colorimetric assay to determine EC50 values.

Results are shown below in Table 9.

TABLE 9 ADC assays Clone ADC EC50 (pM) M26 90.23 M31 34.28 G4 44.35 M220.95 M5 149.5 M29 91.39 B10 54.72 D11 15.85 E3 13.37 G2 28.23 G6 34.07G10 27.94 G12 19.43 G26 34.86 G30 29.33

F. Example 6: Complement Dependent Cytotoxicity (CDC) Assays

Complement dependent cytotoxicity assays were carried out on primarycells from AML patients. Primary AML cells were incubated with CLL1antibodies at various concentrations for 2 hours at 37 C in the presenceof complement. Cell viability was determined by colorimetric Cellglowassay (Promega).

FIGS. 1A-1C shows results from 3 AML patient samples. CLL-1 antibodyclone M26 has an EC50 of about 10-100 ng/mL with these cell samples.FIG. 1C, representing AML sample #52, also shows the effect of clone M31compared to E12 (unrelated mAb) and IgG control.

Results from another round of CDC assays, using 10 ug/mL antibody, areshown in Table 10.

TABLE 10 CDC assays Clone CDC (% killing) M26 17.18 M31 12.14 M5 17.87M22 14.49 D11 18.52

The data show that the CLL-1 antibody clones have significant CDCactivity on primary AML patient samples. The CLL-1 antibody clones areeffective across at least a 5-fold difference in CLL-1 antigen densityin patient samples.

G. Example 7: In Vivo Inhibition of AML Tumor Growth

Two sets of in vivo efficacy studies were carried out. The first was asubcutaneous (SC) tumor engraftment and growth model utilizing the CLL-1positive HL60 AML human cell line in mice. The second was an orthotopic(bone marrow, blood, spleen and lymph node) tumor engraftment andoutgrowth model utilizing the CLL-1 positive OCI AML-5 human AML cellline.

The SC HL60 study was carried out as follows. One of 4 CLL-1 antibodyclones (M5, M13, M26, and M31), or an IgG control, were administeredi.p. at a dose of 200 ug/animal approximately 24 hours prior to SCinoculation of 5×10⁶ or 10⁷ HL60 cells. Animals received additionalantibody doses once per week for the next 6 weeks. The study terminated45 days after the HL60 cell administration. FIG. 2 shows efficacy curvesfor various CLL1 antibody clones (M5, M13, M26, and M31) compared tocontrol.

The OCI AML-5 cell orthotopic studies were carried out as follows.Immunodeficient NSG mice were split into 5 groups of 6 animals/group.One of 4 CLL-1 antibody clones (M5, M13, M26, and M31), or an IgGcontrol, were administered i.p. at a dose of 200 ug/animal approximately24 hours prior to intravenous inoculation of 5×10⁶ or 10⁷ OCI AML-5cells. Animals then received additional antibody doses twice per weekfor the next 2 weeks. The study terminated 4 weeks after administrationof the OCI AML-5 cells. FIG. 3 shows that the CLL-1 antibody clonesdramatically reduced the number of OCI AML-5 cells (labeled hCD45+CSC-030+ and AML CSC030+) in vivo.

H. Example 8: CLL-1 Antibodies are Specific for AML Stem Cells in ADCAssays

The M26 CLL-1 antibody was tested for specific killing in an ADC assay,conjugated to saporin. Primary patient AML cells or normal CD34 positivehematopoietic stem cells isolated from the bone marrow of humansubjects, were seeded into a soft agar colony formation assay (100,000cells/plate). The cells were then incubated in the presence ofCLL-1-saporin toxin-conjugated monoclonal antibody clone M26 for 14days. As shown in FIGS. 4A-4B, the CLL-1 antibody—saporin conjugatecaused selective, specific inhibition of AML stem cell clonogenicgrowth, while normal HSCs were not affectted. The negative controls wereuntreated or treated with an unrelated IgG-saporin conjugate. Theresults demonstrate that CLL-1 antibody conjugated to cytotoxin reducesAML cell colony formation by about 80%, without inhibiting HSC colonyformation. The results indicate that the presently disclosed CLL-1antibodies can be used safely therapeutically to specifically targetCLL-1 expressed on AML cells.

I. Example 9: Human Chimeric CLL-1 Antibodies Bind Human PeripheralBlood Mononuclear Cells (PBMCs) Similar to Mouse CLL-1 Antibody Clones

The variable regions (Fab) of CLL-1 antibody clones M26, M31, and G4were used to make chimeric antibodies with a constant region (Fc) from ahuman IgG1. These human chimeric antibodies are referred to as ChiM26,ChiM31, and ChiG4 (or Chi31G4). To test the specificity of the humanchimeric antibodies compared to the parent mouse antibodies, theantibodies were used to stain different populations of human PBMCs.PBMCs were obtained from two human donors, separated by Ficoll gradient,and pooled. About 2×10⁵ mononuclear cells were blocked with 3% humanserum and then stained with antibodies specific for lineage markers CD89(granulocyte), CD14 (monocyte and granulocyte), CD3 (lymphoid), and CD19(B cell). FIG. 5 shows the FACS results for live-gated cells. The humanchimeric CLL-1 antibodies stain the same myeloid lineage populations asthe mouse CLL-1 antibodies.

J. Example 10: Human Chimeric CLL-1 Antibodies Bind Cynomolgus PBMCsSimilar to Mouse CLL-1 Antibody Clones

To test the specificity of the human chimeric antibodies compared to theparent mouse antibodies, the antibodies were used to stain differentpopulations of cynomolgus PBMCs. PBMCs were obtained from three donors,separated by Ficoll gradient, and pooled. About 2×10⁵ mononuclear cellswere blocked with 3% human serum and then stained with antibodiesspecific for lineage markers CD3 (lymphoid), CD19 (B cell), CD14(granulocyte), CD14 (monocyte), and CD89 (granulocyte). FIG. 6 shows theFACS results for live-gated cells. The human chimeric CLL-1 antibodiesstain the same myeloid lineage populations as the mouse CLL-1antibodies.

K. Example 11: Human Chimeric CLL-1 Antibodies have In VitroAntibody-Drug Conjugate Activity

The ability of the human chimeric CLL-1 antibodies to internalize andmediate ADC was tested on CLL-1 expressing 293 cells in vitro. Cellswere contacted with the indicated antibodies at various concentrations.Matching IgG isotype antibodies were used for negative controls. Thensaporin conjugated secondary antibody (Mousezap® or Humzap®) was addedat a 2:1 ratio, and the cells were incubated for 72 hours. CellTitre-Glo® was added to each culture well and mixed for 5-10 minutes anddetected on a luminescent plate reader. Cell viability was determined byluminescent signal. FIGS. 7A-7B show that the human chimeric CLL-1antibodies (7B) have almost identical ADC activity as the mouse CLL-1antibody clones (7A).

L. Example 12: Human Chimeric CLL-1 Antibodies Mediate AntibodyDependent Cell-Mediated Cytotoxicity (ADCC) Activity

The ability of the human chimeric CLL-1 antibodies ChiM26, ChiM31, andChiG4 (Chi31G4) to mediate ADCC was determined on CLL-1 expressing 293cells. Target cells were added to 96 round bottom wells and incubatedwith the indicated antibodies at various concentrations and effectorcells (Promega®) for 6 hours at 37 C. Viable cells were detected usingPromega ADCC Reporter Assay®. Results are shown in FIG. 8. The human IgGisotype control had no detectable activity, while EC₅₀ in ng/ml forChiM26, ChiM31, and Chi31G4 was determined to be 79, 143, and 105,respectively.

M. Example 13: In Vivo Inhibition of AML Tumor Growth

Two sets of in vivo xenograft studies were carried out with mouse andhuman chimeric CLL-1 antibodies. Both studies utilized NOD/SCID miceirradiated 1 day before tail vein injection with human AML cells on Day0. Both studies included 8 antibody injections over the course of about3 weeks, followed by detection of tumor growth in bone marrow cells.

In the first study, mice were separated into three groups of 6 miceeach: (1) human IgG isotype control; (2) M26; and (3) ChiM31. Mice wereinjected with 3×10⁶ HL60 cells on Day 0. Antibody was administered at200 ug/mouse on Days 1, 4, 7, 10, 13, 16, 19, and 22. Mice weresacrificed on Day 26. Results are shown in FIGS. 9A-9B. FIG. 9A showsthat the CLL-1 antibodies significantly reduced the percentage ofhuCD45+CD33+ AML cells, and FIG. 9B shows that the CLL-1 antibodiessignificantly reduced the percentage of huCD45+ CLL-1+ AML CSCs.

In the second study, mice were separated into five groups of 6 miceeach: (1) human IgG isotype control; (2) M26; (3) ChiM26; (4) ChiM31;and (5) ChiG4. Mice were injected with 5×10⁶ OCI AML-5 cells on Day 0.Antibody was administered at 200 ug/mouse on Days 1, 4, 7, 10, 13, 16,19, and 24. Mice were sacrificed on Day 28. Results are shown in FIGS.10A-10B. FIG. 10A shows that the CLL-1 antibodies apparently eliminatedhuCD45+CD33+ AML cells. A log 10 scale was used to better resolve theresults, as shown in FIG. 10B.

What is claimed is:
 1. An isolated antibody that specifically binds theextracellular domain of human C-type lectin like molecule 1 (CLL-1),wherein the antibody binds a polypeptide consisting of the C-lectindomain of human CLL-1 with a Kd at least 5-fold higher than apolypeptide consisting of the C-lectin and stalk domains of human CLL-1.2. The isolated antibody of claim 1, wherein the antibody binds human orcynomolgus CLL-1 with a Kd of 1000 pM or lower.
 3. The isolated antibodyof claim 1, wherein the antibody binds at least 50% of cells in a sampleof acute myelogenous leukemia (AML) cells from an individual with AML.4. The isolated antibody of claim 1, wherein the antibody bindsquiescent CLL-1 expressing cells.
 5. The isolated antibody of claim 1,wherein the antibody is humanized.
 6. The isolated antibody of claim 1,wherein the antibody is an Fv antibody fragment.
 7. The isolatedantibody of claim 1 conjugated to a therapeutic compound.
 8. Theisolated antibody of claim 1 conjugated to a detectable moiety.
 9. Amethod of determining whether a cell expresses C-type lectin likemolecule 1 (CLL-1), comprising, contacting the antibody of claim 9 withthe cell; and detecting the binding of the antibody to the cell, whereinbinding of the antibody to the cell indicates that the cell expressesCLL-1; and determining whether the cell expresses CLL-1.
 10. The methodof claim 9, wherein the cell is in a biological sample from anindividual that includes hematopoietic cells.
 11. The method of claim 9,further comprising determining whether the cell expresses CD34 or CD38.12. A method for inhibiting survival of a cell expressing C-type lectinlike molecule 1 (CLL-1), comprising contacting the antibody of claim 1with the cell, thereby inhibiting survival of the cell.
 13. The methodof claim 12, wherein the contacting comprises administering the antibodyto an individual, and the cell is in the individual.
 14. The method ofclaim 13, wherein the individual has been diagnosed with amyeloproliferative disorder.
 15. The method of claim 12, wherein themyeloproliferative disorder is selected from the group consisting of:acute myelogenous leukemia (AML), chronic myelogenous leukemia (CIVIL),chronic myelomonocytic leukemia (CMML), myelodisplastic syndrome (MDS),multiple myeloma, plasmacytoma, and myelofibrosis.
 16. A pharmaceuticalcomposition comprising the isolated antibody of claim 1 and apharmaceutically acceptable carrier.
 17. A method of treating amyeloproliferative disorder in an individual, comprising administeringto the individual the pharmaceutical composition of claim 17, therebytreating AML in the individual.
 18. The method of claim 17, wherein theantibody is conjugated to a therapeutic compound.
 19. The method ofclaim 17, wherein the individual has been diagnosed with amyeloproliferative disorder or has undergone therapy for amyeloproliferative disorder.
 20. The method of claim 17, wherein themyeloproliferative disorder is selected from the group consisting of:acute myelogenous leukemia (AML), chronic myelogenous leukemia (CIVIL),chronic myelomonocytic leukemia (CMML), myelodisplastic syndrome (MDS),multiple myeloma, plasmacytoma, and myelofibrosis.